(Boschke F.L.) Medicinal Chemistry

2
Topics in Current Chemistry

Fortschritte der Chemischen Forschung
Medicinal Chemistry
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Contents
Modes of Action of Antimicrobial Agents

Fred E. Hahn
Ansamycins: Chemistry, Biosynthesis and Biological Activity

Walter Wehrli
21
Syntheses and Activity of Heteroprostanoids

Dieter Orth and Hans-Eckart Radunz
51
Hypolipidaemic Aryloxyacetic Acids

Erich Schacht
99
Tilorone Hydrochloride: The Drug Profile

Prakash Chandra and George J. Wright
Author Index Volumes 26-72
125
149
Editorial Board:
Prof. Dr. Michael J. S. Dewar
Department of Chemistry, The University of Texas

Austin, TX 78712, USA
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Sendai, Japan 980
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Institut de Chimie, Universit6 de Strasbourg, 4, rue

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University of Kentucky, College of Arts and Sciences

Department of Chemistry, Lexington, KY 40506, USA
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Institut fiir Physikalische Chemie der Universit~it

Im Neuenheimer Feld 7, D-6900 Heidelberg 1
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Modes of Action of Antimicrobiai Agents
P r o f e s s o r D r . F r e d E. H a h n
Walter Reed Army Institute of Research, Washington, D.C. 20012, U.S.A.
Table of Contents
lI.
Introduction . . . . . . . . . . . . . . . . . . . . .
What Constitutes the Mode o f Action? . . . . . . . . . . . .
HI. The Test Organisms and Drag Effects U p o n Them
. . . . . . . .
IV. The Strategy of Mode of Action Studies

. . . . . . . . . . .
6
1. Inhibition o f DNA Synthesis . . . . . . . . . . . . . .
7
2. Inhibition o f RNA Synthesis . . . . . . . . . . . . . .
9
3. Inhibition o f Protein Synthesis . . . . . . . . . . . . . .
10
4. Inhibition o f Cell Wall Biosynthesis . . . . . . . . . . . .
12
5. Interference with the Structural and Functional Integrity o f Membranes 13
V~ Research on Mechanisms of Action
. . . . . . . . . . . . .
16
VI. Conclusions . . . . . . . . . . . . . . . . . . . . .
17
VII. References
18
F. E. Hahn
I. I n t r o d u c t i o n
Actions of chemotherapeutic drugs have been investigated for more than three
decades. The bibliography of source articles in this research field comprises more
than 10,000 original papers; a number of textbooks and monograph collections have
been published 17). The purpose of this present article can not be an attempt at
reviewing even a small part of the large literature on modes and mechanisms of
action of chemotherapeutic drugs. What is intended, instead, is to present some of the
conceptual outlines and fundamental considerations which have shaped and advanced
this field of science.
When the author of this essay was confronted in 1949 with the problem of elucidating the mode of action of chloramphenicol, there existed virtually no methodological or conceptual guidelines which could be applied to this task. A review on the
mode of action of sulfonamides in 19438) had occupied 87 pages, cited 291 references and did not come to any clearcut explanation of what is considered today one
of the better understood modes of chemotherapeutic drug action.
The reason for these initial difficulties were clearly that scientists did not know
conceptually what to look for, nor methodologically how to look. A review of the
mechanism of action of the tetracyclines, as late as 1967, offered 6 pages of tabulation of "activities" which had been found to be inhibited by these antibiotics 9)
While it is not unusual to read that the advances in the last 15 years are methodological in nature, it is not always appreciated that answers began to come forth only
after investigators had learned to ask pertinent questions.
II. W h a t C o n s t i t u t e s t h e M o d e o f A c t i o n ?
It is appropriate, therefore, to reprint here the postulates of this author which were
offered in a symposium lecture on Modes of Action of Antibiotics at the Fourth
International Congress of Biochemistry in 1958 ~o)
"The problem o f elucidating the mode o f growth inhibition o f an antibiotic is. . . . .
not simply one o f discovering some action upon some physiological process or
biochemical reaction. Many such findings come up during extended investigations
and are not infrequently misinterpreted as modes o f action. The real problem is
conceptual, viz., elimination o f unimportant or secondary effects and identification
o f the primary process or reaction whose inhibition is origirially responsible for
growth inhibition."
"We have adopted a set o f criteria which a physiological process or biochemical
reaction must fulfill in order to be considered the key process whose inhibition leads
to the overall result o f growth inhibition.
1. The inhibited reaction must be o f vital necessity for the economy o f the microbial
cell
2. The inhibition must be produced specifically in organisms whose growth is susceptible to the action o f the drug.

3. The inhibition must be produced by an antibiotic concentration that is o f the
same order as the growth.inhibitory concentration range.
4. The degree o f inhibition must approach an all-or-none effect.
5. The inhibition must depend upon the specific chemical structure o f the antibiotic molecule in precisely the same manner as does the growth-inhibitory effect. "'
Here are some selected examples of research studies which did not consider the
above postulates. In 1949 a paper was published which reported inhibitions of
bacterial esterases by chloramphenicol 1~). No thought was given to the requirement
that the inhibited reaction must be vitally important to growth of susceptible cells.
One year later appeared a paper with the title, on the mechanism of action of
aureomycin t2). Here the effect of chlortetracycline was studied in a mammalian
liver mitochondrial system in which the drug interfered with oxidative phosphorylation.
It was completely missed that liver cells are not target organisms for the antibiotic
action of tetracyclines. This violates the second postulate, viz., the inhibition must
be produced specifically in organisms whose growth is susceptible to the drug under
study. Within a short time, it was shown 13.14)that chlortetracycline binds Mg 2+by
chelation and that the "inhibition" of oxidative phosphorylation was the simple
result of magnesium deficiency. Even recently, a paper was published which reported
effects of the antimalarials, chloroquine and primaquine, on polypeptide synthesis
in a cell-free system from rat liver is)
One of the most frequently neglected rules in the investigation of modes of action
pertains to the use of excessive drug concentrations. In a study of the effects of
chlortetracycline on a (mammalian) D-amino acid oxidase 16), the antibiotic concentration was 1.2 x 10-3M which is 575 tag/ml, re. more than two orders of
magnitude higher than the growth-inhibitory concentrations for susceptible bacteria.
The literature is replete with reports of minor inhibitions of some biochemical
or physiological process by chemotherapeutic drugs. In my own laboratory it was
found that quinacrine caused a slight inhibition of protein biosynthesis in susceptible
bacteria 17) which subsequently was verified in a cell-free poly U system, polymerizing
phenylalanine 18). Since it was also established, however, that the drug is a strong
inhibitor of DNA biosynthesis 17) and that this effect accounts for the bactericidal
action of quinacrine, the slight effect on protein biosynthesis was not mistaken as
the mode of action.
An interesting body of investigations was published on the effects of chlortetracycline on bacterial nitro-reductases (reviewed in9)). Since a reductase enzyme isolated
from chlortetracycline-resistant bacteria was less sensitive to the antibiotic, one could
have hoped that these studies were in some manner directed towards the mode of
action of chlortetracycline. However, antibiotically inactive degradation products
of chlortetracycline also inhibited nitro-reductases from bacteria 19). This was in
disagreement with the last postulate that the observed inhibition must depend upon
the specific chemical structure of the antibiotic molecule in precisely the same
manner as does the growth-inhibitory effect. In our extensive studies on the mode
of action of chloramphenicol, we carried out routinely control experiments with
the antibiotically non-active enantiomer of the drug in order to disallow non-specific
effects which conceivably might have been followed up.
3
F. E. Hahn
An interesting exception to the absolute validity of the tifth postulate is the
considerable activity of chloramphenicol derivatives in cell-free model systems of
protein synthesis when these derivatives are substituted with amino acyl residues
instead of with dichloroacetyl as is the antibiotic itself (rev. in 20)). This has been
traced to the necessity o f the dichloroacetyl grouping in aiding in the permeation
of the antibiotic through the bacterial envelope 21). The amino acyl derivatives have
very low antibacterial activity 20). Permeation failures of actinomycin D, macrolides
and distamycin A with respect to certain families of bacteria occlude the action of
these antibiotics on their intraceUular drug receptors and target reactions but can be
overcome experimentally by measures which render test organisms permeable.
III. T e s t Organisms and Drug E f f e c t s U p o n T h e m

Since quantitative drug actions on the viability and/or growth of microorganisms are
the basic reference parameters of all mode of action studies, the selection of a test
organism and the determination of the effects of a drug upon it are the essential
first steps in all investigations on modes and mechanisms of chemotherapeutic drugs.
If the drug under study is a reasonably broad spectrum antibacterial agent, it is
easy to select a non-pathogenic test bacterium which lends itself to extensive
laboratory investigations. Many such studies have therefore been carried out with
prototrophic strains ofEscherichia coli which grow in mineral medium supplied
with a source of carbon. With a view to anticipated testing for the incorporation of
building blocks into the macromolecules of a test organism, it can be advantageous
to select auxotrophic mutants which may require a certain amino acid (such as
phenylalanine) or thymine for growth. A frequently used test strain is E. coli TAUwhich requires thymine, uracil and arginine.
Chemotherapeutic drugs with an extremely narrow spectrum of activity such as
isoniazid or p.aminosalicylic acid which act exclusively on Mycobacteria are difficult to study, because no convenient test organism is available.
A special problem arose with respect to the antiplasmodial drugs chloroquine
(Resochin) and quinacrine (Atebrin). Plasmodia can not be propagated in cell-free
lifeless media and the limited availability of erythrocytic plasmodial cultures severely
limits biochemical or molecular pharmacological studies on the action of antimalarials.
The actions of chloroquine and quinacrine were, therefore, studied in Bacillus
megaterium or E. coli, respectively 22)
The selection of bacteria as test organisms for the investigation of antimalarial
drugs did, at first, not go unchallenged and required a scientific justification which
was given as follows 22).
"Owing to the near universality of the molecular processes, fundamental to
microbial growth and replication, the choice of test organisms or cell-free test
systems is dictated not so much by the pathogenic properties of specific microorganisms but rather by considerations of methodological feasibility and of the
opportunity of physical and conceptual isolation of a given phenomenon to be
studied. In no instance in which a problem of the mechanism [of action] of a
chemotherapeutic drug has been solved, was such a solution accomplished through
work on a ma]or pathogenic target organism or has required that the study be
extended to such pathogens. In this respect the investigative perspective in molecular
pharmacology differs from that in pathology, immunology or medical microbiology
which must be concerned with the pathogenic, immunogenic or diagnostic properties
of specific etiological agents of communicable diseases. ""
"lt wouM be erroneous to consider an investigation o f the inhibition of major
biosynthetic pathways or of the mechanistic details of such inhibitions merely a
"model" until such a time when a confirmatory analogy study was carried out with
intact of fractionated cells of a pathogenic microorganism for no deeper reason than
that the given chemotherapeutic molecule can be used to cure clinical illness produced
by the growth and replication of such an organism. Essentially, any microorganism
that is subject to growth inhibition by critical concentrations of a given chemotherapeutic drug is, for that very reason, a suitable test organism in mode of action studies."
This reasonig has been fully justified in the sense that subsequent studies on the
action of chloroquine on plasmodia have confirmed the results of preceding work 22)
which used bacteria as test organisms (rev.23)). There are instances, however, in which
no suitable test organism can be found despite a systematic search for it. In the
laboratory of this writer, no test bacterium was discovered which was susceptible
to reasonably low concentrations of quinine and certain new investigational anti.
malarial drugs could not be studied in bacterial cultures because of their limited
solubility in aqueous media.
Among the many microbiological methods of demonstrating growth inhibitions
stands out the determination of decreases in growth rates by graded drug concentrations. Growth rates of cultures which have entered the exponential phase of
multiplication (measured either turbidimetrically, by electronic counting such as in
a Coulter Counter, or by plating and colony counts) are systematically reduced,
yielding families of growth curves with decreasing slopes. The regression coefficients
of such exponential growth curves can be expressed in per cents of inhibition by
(logarithmically) graded drug concentrations, and these percentages, converted to
their probits, are linear functions of the logarithm of drug concentration and permit
interpolation to the 50 per cent inhibitory concentration, IDso ' which is the most
precise measure of antibacterial potency 24). Furthermore, the IDso in molar concentration represents the dissociation constant of a drug-receptor complex which is
formed and its reciprocal value is the apparent association constant. This does not
specify if the receptor is on the cell surface where its occupancy precedes entry of
the drug into the cell or is intracellular and causally responsible for the molecular
mechanism of action of the drug.
This type of kinetic analysis of growth inhibition works with predominantly
bacteriostatic drugs such as chloramphenicol or the tetracyclines25) but has been
extended to the study of the bactericidal antibiotic, streptomycin 26).
Brought to logical conclusion, it was shown that the probit transformations of
bacterial growth inhibition and inhibition of DNA biosynthesis by Nitroakridin
3582 were superposable while the same functions for inhibition of RNA and protein
biosyntheses were superposable upon each other but indicated a lesser susceptibility
of the test organism. This led to the conclusion that the mode of antibacterial action
of the nitroacridine was its inhibition of DNA biosynthesis 27).
F. E. Hahn
Kinetic analysis of growth inhibitions by graded concentrations of a chemotherapeutic drug failed with penicillin 2s). Up to a certain threshold drug concentration
and for a fraction of one doubling time, the antibiotic had little influence (turbidimetrically) on growth rates; beyond these critical time and concentration limits,
the test culture underwent rapid lysis, i.e. morphological destruction of the bacterial
cells. The same problem can logically be predicted for all drugs which interfere with
the integrity of the cell wall, resulting in lysis and physical disassembly of the test
ceils.
The concentration of choice for mode of action studies is the lowest drug concentration which inhibits growth entirely. This can either be estimated by extrapolation of the probit-transformed log dosage response correlation or by determination
of the MIC, Le. minimal inhibitory concentration, by the method of serial twofold
dilution of drug-containing medium in test tubes, inoculation of these media and
visual observation of growth after incubation overnight.
IV. T h e S t r a t e g y o f M o d e o f A c t i o n Studies
At the early beginning of mode of action studies, the field was without a systematic
approach and, hence, relegated to empirical inquiry in which scores of enzyme
reactions and physiological processes had to be tested for susceptibility to a chemotherapeutic drug under investigation in the hope that some major effect would be
uncovered which could be considered significant. An early review on sulfonamides s)
and a long tabulation of actions of tetracyclines9) are indicative of the results which
were empirically obtained.
A review of the mode of action field today leads to the conclusion that there
exists a very linited number of interferences with physiological processes of microbial ceils which result in cell death or in inhibitions of cellular replication. Regardless
of the underlying mechanistic reasons, there are mainly five categories of action
which lead to chemotherapeutic potency.
1. Inhibition of DNA synthesis,
2. inhibition of RNA synthesis,
3. inhibition of protein synthesis,
4. inhibition of cell wall synthesis and
5. interference with the structural and functional integrity of cell membranes.
Substances such as the antibiotics of the antimycin family which interfere with
electron transport in the respiratory chain are generally toxic and, for this reason,
unsuited as chemotherapeutic drugs. The only practical use of antimycin is that as
a fish poison.
After the selection of a suitable test organism, the strategy of mode of action
studies involves basically the testing of the compound under study for its effects
on each of the five processes listed above. This requires invariably the study of
actively growing cultures or, better, of organisms which are incubated in a medium
which supports growth and multiplication. Bactericidal drugs, for example, penicillin
and streptomycin have no lethal effects on non-growing bacterial suspensions and

actions on macromolecular syntheses can, of course, not be investigated in "resting"
organisms, i.e. in suspensions of bacteria in buffered salt solutions devoid of nutrients.
The resting cell technique was mostly used in manometric experiments on gas
exchanges in cell suspensions, oxidizing a variety of substrates 28).
1. Inhibition o f DNA Synthesis is usually signalled by bactericidal actions of the
drug under investigation: organisms which can not replicate their chromosomes
cannot produce a viable progeny. In addition, inhibitions of DNA synthesis frequently
give rise to long filamentous forms of bacteria. It is believed that the completion of
one round of chromosomal DNA biosynthesis produces a "signal" which actuates
cross septation and cytokinesis. In its absence, the cells grow longer but do not divide.
Figure 1 shows such filaments ofE. coli after exposure to 2 10 -4 M quinacrine for
24 hours 17) and Fig. 2 shows another microscopic field photographed in the fluorescence microscope and demonstrating the fluorescent staining of the bacterial
filaments by the drug.
Fig. 1. Filaments ofEscherichia coli, formed during 24 hours o f growth in the presence of
9
9 17)
2 x 10 - 4 M qumacrme
One classical and early example of selective inhibition of DNA biosynthesis is

shown in Fig. 3 for the anitbiotic, mitomycin C 29). A concentration of 0.1/ag/ml
(3 x 10-7 M) completely inhibited DNA synthesis in E. coli B, while RNA synthesis,
protein synthesis and "growth" meaning turbidity, i.e. cell mass increase, continued.
However, after the experimental period of only 90 min, the number of viable bacteria
had decreased by 85 per cent. By that time, bacterial filaments were visible under
the microscope.
With the introduction of radiochemical methods, DNA biosynthesis and its
inhibition is usually followed either by measuring the incorporation of radioactive
thymine into thymine auxotrophs of bacteria or the incorporation of radioactive
thymidine into prototrophic organisms. In the latter instance, it is practical to include
in the experimental medium a large excess of non-radioactive deoxyadenosine in order
F. E. Hahn
Fig. 2. Same object as in Fig. 1 but photographed under the fluorescence microscope
0.30
Control
- .....
/~/~
MC 0.1 p g / m l
/~/"
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0
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"" ~ " ! J ' "
j~--
,~o'~"
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./.~
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z
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i -
- o 06
->"
J004
,<
__x ............
l
i
30
60
Zncubation time in minutes
~_
O.02 o
II 0
90
Fig. 3. Selective inhibition of DNA biosynthesis in E. colt by 0,1 pg/ml of mitomycin C 29)

to decrease the hydrolysis of the labelled precursor by a deoxynucleosidase which
bacteria excrete into their growth medium. Without this precaution the labelled
compound in the medium is quickly dissipated and incorporation levels off owing
to an exhaustion of thymidine.
A large number of antibiotic and synthetic inhibitors of DNA biosynthesis form
complexes with DNA and act as template poisons for the replication of chromosomal
DNA 6, 7). These substances are preferential inhibitors of eucaryotic cells such as
protozoa or neoplastic cells. The only antibacterial chemotherapeutic drug which
is a selective inhibitor of DNA biosynthesis is nalidixic acid. This synthetic compound does not form complexes with DNA but acts in an unknown manner which
appears to involve a perturbation of the interaction of DNA with a bacterial membrane
system which is instrumental in DNA replication 3~
In bacteria, DNA complexing drugs or experimental DNA complexers have been
found selectively to inhibit the replication of plasmidic, especially of R-factor, DNAs
and, hence, to eliminate resistance determinants and restore antibiotic sensitivity to
such organisms 31). This has opened the prospect of developing a special category of
drugs for the combination (with antibiotics) treatment of Gram-negative bacterial
infections 32).
2. Inhibition o f RNA Synthesis usually has bacteriostatic consequences since it
does not result in severely unbalanced biosynthesis and growth. When RNA biosynthesis is turned off by an inhibitor, the cell cannot replenish messenger RNA which,
in microorganisms, is subject to rapid turnover. The progressive deficiency in
messenger RNA results in a progressive decay in the rate of protein biosynthesis.
For example, in cultures of Bacillus subtilis, protein synthesis had ceased entirely
after one fourth of a doubling time when RNA synthesis was specifically and
completely inhibited by actionomycin D 33) . Since the initiation of a new round of
DNA biosynthesis requires the ad hoc synthesis of "initiator protein(s)," DNA
biosynthesis comes to a standstill after a lag as the result of the failure of initiator
protein synthesis. This interesting sequence of events was first described by Kirk 34)
for the action of actinomycin D in Staphylococcus aureus.
Actinomycin D is a selective inhibitor of RNA synthesis but its use in cancer
chemotherapy is severely limited owing to its toxicity. It is thought that the two
cyclic peptide moieties of the antibiotic whose three-membered aromatic ring system
is intercalated into doublestranded DNA occlude the minor groove of duplex DNA
and impede the progression of the enzyme, RNA polymerase.
An important family of inhibitors of procaryotic RNA synthesis are the antibiotics
of the ansamycin series whose prototype is rifampicin. It inhibits bacterial RNA
synthesis at the extremely low concentration of 0.01/ag/ml (1.4 10 -8 M) by binding
to bacterial RNA polymerase as).
With the exceptions of mitomycin C and actinomycin D which are, at growthinhibitory concentrations, specific inhibitors of DNA or RNA biosyntheses, other
DNA-complexing chemotherapeutic drugs inhibit both categories of biosyntheses
although usually to different extents which depend not only on the drug concentrations but also on the test organism. For example, quinacrine is a preferential
inhibitor of DNA biosynthesis in E. coli 17), while in Bacillus cereus the drug acted
F. E. Hahn
predominantly on RNA biosynthesis 36). While such species-dependent specificities
in the actions of DNA-complexing drugs are fairly generally recognized, the underlying
reasons are not clear. In in vitro studies on inhibitions of DNA and RNA polymerase
reactions by quinacrine, the two dosage response correlations were not significantly
different from each other 22).
3. Inhibition o f Protein Synthesis is a most predominant mode of action among
antibiotics. There are more than 20 single antibiotics or congeneric families of
antibiotics which act in this manner. With the exception of the aminoglycosides which
are bactericidal drugs, inhibitions of protein synthesis produce bacteriostasis. Interestingly, there is no synthetic compound among clinical chemotherapeutic drugs
which inhibit protein biosynthesis, although a number of experimental amino acid
antimetabolites affect protein synthesis by inhibiting amino acyl transfer RNA
synthetases.
Chloramphenicol and chlortetracycline were the first antibiotics recognized to
be inhibitors of protein synthesis on the basis of findings that they inhibited
induced enzyme syntheses in bacteria after induction had occurred and syntheses
had been under way for considerable periods of time 37).
Inhibitions of protein synthesis were subsequently confirmed by chemical analyses
for protein in growing bacterial cultures 38' 39). Figure 4 39) shows the specific
inhibition of protein biosynthesis by chloramphenicol in E. coli and the continuation
of RNA ("ribose") and DNA biosynthesis for an experimental period of 50 minutes.
Similar results have been typically obtained with other inhibitors of protein biosynthesis.
While the synthesis of DNA levels off for reasons discussed in the preceding
section, RNA synthesis in chloramphenicol-exposed bacteria can continue for
extended periods of time. Some of this excess RNA is transfer RNA or is found
in incomplete (with respect to protein) ribosomelike particles (rev. in4~ but
much of the bacterial RNA, accumulating during inhibition of protein synthesis
is messenger RNA 41). When bacteria are released from inhibition of protein
synthesis, growth does not resume immediately in minmal medium. Instead, there
is considerable messenger RNA breakdown and excretion of its products into the
medium4~, 42) before balanced growth begins again. However, if the antibiotic-free
organisms are resuspended in amino-acids containing media 43' 44), no breakdown
of RNA is observed and the organisms resume protein synthesis without delay, soon
accompanied by RNA synthesis in the manner characteristic of balanced growth 44).
Evidently, the unbalanced synthesis of excess RNA during inhibition of protein
synthesis is not bactericidal.
The "bacteriostatic" effect of chloramphenicol has been investigated quantitatively 4s).
Addition of the antibiotic to cultures ofE. coli B/r growing exponentially in brainheart infusion broth with a generation time of 21.90 min was followed by considerable further increase in total cell number (as measured in a Coulter counter)
and an initial increase in the number of colony-forming bacteria which then
progressively decreased by 75 per cent during continued incubation for a total
of 13.7 generation times (Fig. 5). Division of cells in chloramphenicol-containing
cultures was observed under the microscope 4s). It is possible that "bacteriostasis"
10
Modes o f Action of Antimicrobial Agents

12
60
50
-$
s 30
J"
~ ' - -5"-
o Protein
o
<20
c.
<::L
/~
"/
//
10
~..-
40
50
._..----
/4/
I J r
-Z
20
30
minutes
10
Fig, 4. Selective inhibition of protein biosynthesis in E. coli by 1.9 x 10 - 4 M chloramphenicoi39)
5-10 7
,x
o Total count
Viable count
9 10
60
120
180
Time in minutes
240
300
Fig. 5. Changes ia total and in viable count of E, eoli B/r before and after addition (arrow) of
chloramphenicol to 1.5 x lO - 4 M45)
II
F. E. Hahn
is an oversimplified concept supported by not more than conventional serial
dilution of cultures, followed by plating and colony counting.
Inhibition of protein synthesis by aminoglycoside antibiotics, especially by
streptomycin, is bactericidal (rev.46)). The antibiotic binds to the smaller ribosomal
subunit and leads to the formation of abortive initiation complexes of ribosomes,
streptomycin and amino acyl tRNA which progressively trap ribosomes in the
form of such biologically irreversible complexes. When protein synthesis is
prematurely terminated by puromycin and ribosomes are thus made available for
reinitiation of de novo protein biosynthesis, the bactericidal action of streptomycin
is accelerated 47). Destruction of ribosomes under the influence of primaquine
operationally also results in non-occurrence of protein synthesis and in a marked
bactericidal effect 48' 49)
4. Inhibitions of Cell Wall Biosynthesis. All bactericidal modes of action involve

the alteration or destruction of some component (s) of the cell whose physiological
function is of vital importance and cannot be compensated for or repaired by other
cell constituents. This has been described above for the bacterial chromosome (DNA)
and for the total population of bacterial ribosomes. The most prominent bactericidal
effect, however, is caused by interferences with the biosynthesis of the cell wall
polymer. Such a mode of action was proposed for penicillin on the basis of
morphological observations in 1946 S~
the underlying biochemistry was still
unknown.
"The morphological changes described . . . . . . . in particular the failure of proper
cell division and the ready occurrence of swelling and protoplasmic protrusion
suggest that penicillin interferes specifically with the formation of the outer
supporting cell wall, while otherwise allowing growth to proceed until the organism
finally bursts its defective envelope and so undergoes lysis so). ,,
Such experiments with E. coli were subsequently refined through the introduction
into the medium of sucrose for osmotic protection of the spheroplasts formed, and
the sequence of events during the action of penicillin was photographed under the
phase contrast microscope (Fig. 6) 51).
After Park isolated and identified uridine-diphosphate-peptidoglycan products
that accumulate to high concentrations in penicillin-exposed Staphylococcus aureus s2),
it took the results of several years of independent investigations on the chemical
composition of bacterial cell walls to recognize that the products were biosynthetic
precursors of the cell wall polymer sa). The biochemistry of bacterial cell wall synthesis
was subsequently studied in great detail by Strominger and his associates with the
conclusion that penicillins interfere with a final cross- linking step in the assembly
of the cell wall polymer (rev. 54)). The morphological manifestation of bacterial
destruction in Fig. 6 are, hence, the results of a defective growth of the cell wall
which weakens this structure to an extent that it can no longer withstand the
internal osmotic pressure. Disruption and lysis of Gram-positive bacteria under the
influence of penicillin has also been demonstrated ss) although the morphology of
these events is not so dramatic as that in Gram-negative bacteria (Fig. 6).
Additional antibiotics which interfere with cell wall synthesis are the cephalosporins whose mode of actionis similar to that of penicillins, cycloserine which
12
Modes of Action of Antimicrobiat Agents
Fig. 6. Sequential phases of

penicillin-induced fromation of
spheroplasts of E. coli B as photographed under the phase contrast
microscopes 1)
inhibits the formation of the D-alanyl-D-alanine moiety of the peptidoglycan (rev. s6))
and phosphonomycin which acts on an earlier step of peptidoglycan synthesis as an
antagonist of phosphoenolpyruvate sT)
A synthetic inhibitor of cell wall synthesis is 3-fluoro-D-alanine which inhibits
bacterial growth in competition with D-alanine and exhibits the "chemotherapeutic
paradox" of being more active in vivo than in vitro 5a) .
5. Interference with the Structural and Functional Integrity o f Membranes. The
membrane is another structure whose alteration of partial destruction has a bactericidal
effect. Its vital importance to the economy of the bacterial cell is that it controls and
mediates permeation, i.e. it determines which chemicals enter or leave the cell and
which do not. Sterilizing agents such as phenols or detergents and also non-polar
solvents such as toluene affect the lipid constituents of the membranes and cause
chaotic and uncontrolled permeability through which the cell loses much of its
essential biochemicals such as intermediates of synthetic pathways (the metabolic
"pool") and coenzymes into the surrounding medium. This leakage produces
irreversible metabolic starvation .as indicated by the simultaneous failure of all
macromolecular biosynthesesand, hence, is bactericidal.
13
F. E. Hahn
Fig. 7. Fluorescence photomicrograph of Bacillus megaterium after treatment with fluorescent

polymyxin67)
There are relatively few chemotherapeutic drugs which cause permeability
increases in membranes such as the antifungal polyenes and the antibacterial circular
oligopeptides of the tyrocidin, gramicidin and polymyxin families. Most of these are
too toxic for systemic use but polymyxins have been given systemically in severe
Pseudomonas infections with an attending risk of renal toxicity.
Studies on the nature of the antibacterial action of surface-active drugs have been
carried out and/or reviewed by Newton sg). Polymyxin was labelled with a
fluorescent dye without significant decrease in antibiotic potency. Fluorescence
microscopy showed that the antibiotic became associated with the "boundary
structures" of bacteria (Fig. 7). When the cell walls were removed from such bacteria
by treatment with lysozyme, the fluorescent compound was shown to be associated
with the protoplast membrane. Furthermore, the permeability change, induced by
polymyxin was demonstrated by adding N-tolyl-a-naphthylamine-8-sulfonic acid to
polymyxin-treated bacteria. Aqueous solutions of this dye do not fluoresce but when
it reacts with proteins, the conjugates are strongly fluorescent. Suspensions of
Pseudomonas aeruginosa did not take up the dye and, hence, did not become
fluorescent. However, the addition of polymyxin produced immediate bacterial
fluorescence, thereby indicating that the bacteria had been rendered permeable to
the compound and their proteins had reacted with it s9).
A second category of membrane-active antibiotics are the depsipeptides with
valinomycin as the prototype. These substances act as iononophores and increase
permeation of K + ions. The antibacterial effect of 10 - 6 M valinomycin can be reversed
by supplying an excess of K* 60)
Finally, one 2-hydroxy-3-alkyl-1,4-naphthoquinone has been shown to be bactericidal
and to produce a shut-down of all major biosynthetic processes. This is the result of
an inhibition of the transport of nutrients/precursors through the membrane into
the bacterial cell. Detailed experiments on the permeation of uracil demonstrated
that ingress and egress were inhibited by the substituted naphthoquinone 61).
Following this capsule review of the five major categories of modes of action, it
is now possible to outline the strategy of mode of action studies which leads to the
classification of drugs as belonging into one of these categories.
After the selection of a suitable test organism, the first step is to measure the
response of the growth rate to graded concentrations of the drug under study, as
14

outlined in Section III. When this is carried out turbidimetrically, observations of
decreases in absorbance will indicate lysis of the test cells such as can result from
inhibitions of cell wall biosynthesis or from the effects of membrane-active substances.
A second step is to determine if the drug under study is predominantly bacteriostatic or bactericidal. While there will always occur some decrease in the number
of colony-forming cells under the influence of a growth-inhibiting drug, a bactericidal
effect is considered one in which an exponential decrease in the number of viable
cells is two decadic logarithms or greater within one doubling time. When such studies
of viability are carried out, it is not an infrequent occurrence to find a narrow range
of lowest drug concentrations which are merely bacteriostatic. A marked bactericidal
action can signal effects on DNA synthesis, cell wall synthesis or membrane integrity
as underlying causes.
In the special case in which a growth inhibitor has a structure, reminiscent of
an antimetabolite of some metabolite of intermediary metabolism, it can be useful
to compare growth inhibitions in minimal medium with those in broth media. For
example, L-cycloserine, the enantiomer of the antibiotic D-cycloserine, inhibits
bacterial growth only in minimal medium devoid of alanine (unpublished observations). Furthermore, DNA-complexing drugs can be sequestered when DNA is a
constituent of the growth medium 34), and in certain reported instances, the
antagonism of growth inhibitions by enriched media has not been satisfactorily
explained (for example, 62' 63)).
The next, and most important, step is to test the effects of the drug on
macromolecular biosyntheses. For RNA and protein there exist sensitive colorimetric methods but the diphenylamine method for determination of DNA is in
many instances not sensitive enough. It has become customary to follow macromolecular biosyntheses by measuring incorporations of radioactive precursors into the
cellular polymers. Reference has already been made to the use of thymidine (or
thymine) in the study of DNA biosynthesis. The label of choice for following RNA
biosynthesis is uracil. Protein synthesis can be followed by using any of a number
of radioactive amino acids. Care should be taken to avoid glycine which is an
intermediate in a number of biosynthetic pathways or those amino acids (alanine,
glutamic acid, aspartic acid, valine or lysine) whose biosyntheses are not regulated
by the rate of protein synthesis or which also become constituents of the cell wall
polymer. Cell wall biosynthesis can be studied by measuring the incorporation of
a-e-diaminopimelic acid if the wall of the test organism contains this compound
instead of lysine 4a).
Specific inhibitions of DNA or protein biosyntheses have been illustrated in
Figs. 3 and 4. Inhibition of protein synthesis results, after some delay, in the
gradual decay of the rate of DNA synthesis as reviewed earlier in this section. The
more complicated biosynthetic interrelations and consequences of inhibitions of
RNA synthesis have also been reviewed with special reference to the action of
actinomycin D 34). In such instances it is important to concentrate on the early
changes in rates of macromolecular syntheses: after some delay, inhibitions of RNA
synthesis will have produced shutdowns in protein and DNA syntheses.
15
F. E. Itahn
Inhibitions of cell wall biosynthesis can be determined independently from DNA,
RNA and protein biosyntheses. They will usually have been signaled by bactericidal
effects and lysis of the test culture. In many instances, inhibition of cell wall
synthesis results in the progressive accumulation of intermediates which can be assayed
colorimetrically64, 6s) for N-acylaminohexose in acid-soluble fractions of the test
bacteria.
In those instances in which all categories of macromolecular biosyntheses fail
entirely 61), the explanation can be sought either in effects on the bacterial
membrane or in a failure of energy generation or transduction.
It is apparent that the definition of a mode of action in physiological/biochemical
terms can be accomplished by a conventional set of tests for which the strategy has
been outlined in this section. This also applies to substances which interfere with
intermediary metabolism such as the inhibitors of the synthesis or transformations
of folic acid. The task is relatively simple and unambiguous for predominantly
bacteriostatic drugs. However, in the case of a rapid bactericidal effect, late
biosynthetic failures after most cells have lost viability are in the nature of post
mortem findings which must be interpreted with caution. When rapid killing of the
test organism has been found in the preliminary stages of the investigation, attention
must become focused on the earliest effects which can be determined and considered
responsible for the ensuing cell death.
V. R e s e a r c h o n M e c h a n i s m s o f A c t i o n
After the mode of action of a chemotherapeutic drug has been defined and categorized
through the application of the strategy outlined in the preceding section, the
scientific task arises to investigate the underlying mechanism in molecular terms.
Although a very considerable scientific effort has been made and continues in
research on mechanisms of action of antimicrobial and antitumor agents, there
exist relatively few instances in which definitive explanations of drug actions have
been achieved in molecular pharmacological terms.
Even for antimicrobial drugs wich have the same mode of action, the mechanisms
of actions in molecular terms can be quite different. An instructive example of this
is the body of knowledge on actions of inhibitors of protein synthesis at the
ribosomal level 66). Since the mechanistic details of protein biosynthesis as well
as of DNA replication are still incompletely resolved, studies on mechanisms of
action of inhibitors of these biosynthetic processes frequently remain inconclusive
when the target reaction is not known. Each problem of the mechanism of action of
a chemotherapeutic drug becomes an individual research problem in its own right,
after the mode of action has been elucidated in the manner described in this article.
For this reason, a clearcut general strategy of this type of research can not be
formulated. This is especially the case for all antibiotics and synthetic drugs which
have been discovered empirically.
In contrast, substances which have been premeditatively developed as antimetabolites
and possess growth-inhibitory activity for microorganisms, lend themselves to
16

investigation of the antimetabolic relationship for which they have been deliberately
designet. While it may be tempting to review the few instances of successful elucidation of mechanisms of action, no systematic strategic approaches to such investigations would emerge, and such a review of existing knowledge which has accumlated
over the years would have no place in a volume is dedicated to Topics in Current
Chemistry.
VI. C o n c l u s i o n s
This article has traced the development of a strategy for mode of action studies of
chemotherapeutic drugs from its blindfolded empirical beginnings to the current
state in which it is possible to assign a category of mode of action to a given substance within a limited period of investigative time, provided a suitable test organism
can be found. This current research strategy has been described.
Successful solutions to the problems of mechanisms of action which always emerge
after a mode of action has been pinpointed can not yet be organized systematically,
and critical consideration of instances in which such studies have been definitively
successful do not offer much guidance to an investigator who is faced with a new
problem. It will take considerable time until the biochemical process patterns of
microorganisms are known to such an extent that a systematic research strategy can
be developed on the basis of this knowledge.
17
F. E. Hahn
VII. References
1) Newton, B. A., Reynolds, P.E. (eds.): Biochemical studies of antimierobial drugs. Cambridge:
University Press 1966
2) Gottlieb, D., Shaw P.D., (eds.): Antibiotics I. Mechanism of action. Berlin-Heidelberg-New
York: Springer 1967
3) Franklin, T.J., Snow, G.A.: Biochemistry of antimicroblal action. New York: Academic
Press 1971
4) Sch6nfeld, H., DeWeck, A., (eds.): Antibiotics and chemotherapy 17. Mode of action.
Basel: S. Karger 1971
s) Gale, E.F., Cundllffe, E. Reynolds, P.E., Richmond, M.H., Waring, M.J.: The molecular
basis of antibiotic action. London: Wiley 1972
6) Kersten, H., Kersten, W.: Inhibitors of nucleic acid synthesis. New-York-Heidelberg-Berlin:
Springer 1974
7) Corcoran, J.W., Hahn, F.E, (eds.): Antibiotics III. Mechanism of action of antimicrobial and
antitumor agents. Berlin-Heidelberg-NewYork: Springer 1975
8) Henry, R.J.: Bacteriol. Rev. 7, 175 (1943)
9) Laskin, A.I., in: Antibiotics I. Mechanism of action. Berlin-Heidelberg-NewYork: Springer
1967, p. 33I
10) Hahn, F.E.: Proe. Fourth Internat. Congr. Binchem. 5, 104 (1958)
11) Smith, F.N., Worrel, C.S., Swanson, A.I.: J. Bacteriol. 58, 803 (1949)
12) Loomis, W.F.: Science 111,474 (1950)
13) Van Meter, J.C., Oleson, J.J.: Science 113, 273 (1951)
14) Van Meter, J.C., Spector, A., Oleson, J.J., Williams, J.H.: Proc. Soc. Exptl. Biol. Med. 81,
215 (1952)
15) Roskoski, Jr., R., Jaskunas, S.R.: Biochem. Phatmacol. 21,391 (1972)
16) Yagi, K., Okuda, J. Ozawa, T., Okada, K.: Biochim. Biophys. Acta 34, 372 (1959)
17) Ciak, J., Hahn, F.E.: Science 156, 655 (1967)
18) Wolfe, A.D., Hahn, F.E.: Naturwissensch. 59, 277 (1972)
19) Saz, A.K., Weiss Brownell, L., Slide, R.B.: J. Bacteriol. 71,421 (1956)
20) Hahn, F.E., Gund, P., in: Topics in infectious diseases 1. Drug receptor interactions in
antimicroblal chemotherapy. Wien: Springer 1974, p. 245
21) Telesnina, G.N., Novikova, M.A., Zhdanov, G.L., Kolosov, M.N., Shemiakin, M.M.:
Experientia 23, 427 (1964)
22) Hahn, F.E., O'Brien, R.L., Ciak, J., Allison, J.L., Olenick, J.G.: Military Med. 131, 1971
(1966)
23) Hahn, F.E., in: Antibiotics III. Mechanism of action of antimierobial and antitumor agents.
Berlin-Heidelberg-NewYork: Springer 1975, p. 58
24) Hahn, F.E., in: Topics in infectious diseases. Vol. 1. Wien: Springer 1974, p. 3
25) Ciak, J., Hahn, F.E.: J. Bacteriol. 75, 125 (1958)
26) Treffers, H.P.: J. Bacteriol. 72, 108 (1956)
27) Wolfe, A.D., Cook, T.M., Hahn, F.E.: J. Bacteriol. 108, 320 (1971)
28) Umbreit, W.W., Burris, R.H., Stauffer, J.F.: Monometric techniques and related methods
for the study of tissue metabolism. Minneapolis: Burgess 1945
29) Shiba, S., Terawaki, A., Taguchi, T., Kawamata, J.: Biken's Journal 1, 179 (1958)
30) Goss, W.A., Cook, T.M., in: Antibiotics III. Mechanism of action of antimicrobial and
antitumor agents. Berlin-Heidelberg-NewYork: Springer 1975, p. 174
al) Hahn, F.E., Ciak, J.: Antimicrobialagents and chemotherapy. 9, 77 (1976)
32) Hahn, F.E., in: Antibiotics and chemotherapy. Vol. 20, Basel: S. Karger 1976, p. 196
33) Levinthal, C., Keynan, A., Higa, A.: Proc. Nat. Acad. Sci. U.S. 48, 1631 (1962)
34) Kirk, J.: Biochim. Biophys. Acta 42, 167 (1960)
3s) Wehrli, W., Staehelin, M., in: Antibiotics III. Mechanism of action of antimicrobial and
antitumor agents. Heidelberg-New York: Springer 1975, p. 252
36) Seligman, M.L., Mandel, H.G.J. Gen. Microbiol. 68, 135 (1971)
37) Hahn, F.E., Wisseman,C.L.: Proc. Soc. Exp. Biol. Med. 76, 533 (1951)
18
Modes of Action of AntimicrobialAgents

38) Gale, E.F., Folkes, J.P.: Biochem. J. 53, 493 (1953)
39) Wisseman,C.L., Smadel, J.E. Hahn, F.E., Hopps, H.E.: J. Bacteriol. 67, 662 (J 954)
40) Hahn, F.E., in: Antibiotics I. Mechanism of action. Berlin-Heidelberg-NewYork: Springer
1967, p. 308
41) Hahn, F.E., Wolfe, A.D.: Biochem. Biophys. Res. Comm. 6, 464 (1961)
42) Hahn, F.E., Schaechter, M., Ceglowski, W.S., Hopps, H.E., Ciak, J.: Biochim. Biophys. Acta
26, 469 (1957)
43) Horiuchi, T., Sunakawa, S., Mizuno, D.: J. Biochem. (Japan) 45, 875 (1958)
44) Aronson, A.I., Spiegelman, S.: Biochim. Biophys. Acta 53, 84 (1961)
45) Allison, J.L., Hartman, R.E., Hartman, R.S., Wolfe, A.D., Ciak, J., Hahn, F.E.: 1. Bacteriol.
83, 609 (1962)
46) Hahn, F.E., in: Antibiotics and chemotherapy. Vol. 17, Basel: S. Karger 1971, p. 29
47) White, J.R., White, H.L.: Science 146, 772 (1964)
48) Olenick, J.G., Hahn, F.E.: Antimicrobialagents and chemotherapy. 1,259 (1972)
49) Olenick, H.G.: Antimicrobialagents and chemotherapy. 8. 754 (1975)
50) Duguid, J.P.: Edinburgh Med. J. 53, 401 (1946)
51) Hahn, F.E., Ciak, J.: Science 125, 119 (1957)
52) Park, J.T.: J. Biol. Chem. 194, 877 (1952)
53) Park, J.T., Strominger, J. L.: Science 125,99 (1957)
54) Strominger, J.L., Willoughby, E., Kamiryo, T. Blumber, P.B., Yocum, R.R.: Ann. New York
Acad. Sci. 235, 210 (1974)
5s) Ciak, J., Hahn, F.E.: Science 137, 982 (1962)
56) Neuhaus, F.C., in: Antibiotics I. Mechanism of action. Berlin-Heidelberg-NewYork:
Springer 1967, p. 40
57) Kahan, F.M., Kahan, J.S., Cassidy, P.J., Kropp, H.: Ann. New York Aead. Sei. 235, 364 (1974)
58) Kollonitsch, J., Barash, L., Kahan, F.M., Kropp, H.: Nature 243, 346 (1973)
59) Newton, B.A., in: The strategy of chemotherapy. Cambridge: University Press 1958, p. 62
60) Harold, F.M., Baarda, J.R.: J. Bacteriol. 94, 53 (1967)
61) Olenick, J. G., Hahn, F.E.: Ann. New York Aead. Sci. 235, 542 (1974)
62) Wacker, A.: Zschr. Naturforsch. 6B, 173 (1951)
63) Zygmunt, W.A.: Biochem. Biophys. Res. Comm. 6, 324 (1961)
64) Reissig, J.L., Strominger, J.L., Leloir, L.F.: J. Biol. Chem. 217, 959 (1955)
65) Ciak, J., Hahn, F.E.: Antibiotics and chemotherapy. 9, 47 (1959)
66) Weisblum, B., Davies, J.: Bacteriol. Rev. 32, 493 (1968)
67) Gale, E.F.: Synthesis an organization in the bacterial cell. New York: Wiley, 1959, p. 19
Received January 18, 1977
19
Ansamycins
Chemistry, Biosynthesis and Biological Activity
Dr. W a l t e r W e h r l i
Pharmaceuticals Divisions, CIBA-GEIGY LTD, Basel, Switzerland
Table of Contents
Introduction
22
1.
1.1.
1.2.
1.3.
1.4.
1.5.
1.6.
1.7.
Chemistry . . . . . . . . . . . . . . . . . . . . .
22
23
27
28
29
29
29
31
2.
2.1.
2.2.
2.3.
Biosynthesis . . . . . . . . . . . . . . . . . . . .
3.
3.1.
3.1.1.
3.1.2.
3.2.
3.2.1.
3.2.2.
3.2.3.
3.2.4.
3.2.5.
3.3.
3.4.
3.5.
5.
Rifamycins . . . . . . . . . . . .
Streptovaricins . . . . . . . . . . .
Tolypomycin Y . . . . . . . . . . .
Naphthomyc/n . . . . . . . . . . .
Geldanamycin . . . . . . . . . . .
Maytansine and Related Compounds . . . .
Streptolydigin and Tirandamycin . . . . .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Rifamycins . . . . . . . . . . . . . . . . . . . .
Streptovaricins . . . . . . . . . . . . . . . . . . .
Geldanamycin and Maytansine . . . . . . . . . . . . . .
Biological Actions
31
33
33
34
. . . . . . . . . . . . . . . . . .
The Various Modes of Action . . . . . . . . . . . . . .
Effects Produced by Drug Concentrations Below 1 #g/ml ( < 10-6 M)
Effects Produced by Drug Concentrations Over 1/~g/ml ( < 10 - 6 M) .
Effects on Bacteria . . . . . . . . . . . . . . . . . .
Interaction with DNA-dependent RNA Polymerase . . . . . . .
Mode of Action . . . . . . . . . . . . . . . . . . .
Structure-Activity Relations . . . . . . . . . . . . . . .
Nature of Ansamycin Resistance in Mutants
. . . . . . . . .
Other Effects on Bacteria . . . . . . . . . . . . . . . .
Effects o n Eukaryotes . . . . . . . . . . . . . . . . .
Effects o n RNA T u m o u r Viruses . . . . . . . . . . . . .
Effects on DNA Viruses and Larger Infectious Agents such as
Chlamydiae . . . . . . . . . . . . . . . . . . . .
34
35
35
35
36
36
37
37
39
40
40
42
Conclusions and Summary
43
References
. . . . . . . . . . . . . . . . . . . .
42
45
w. Wehrli
Introduction
The ansamycin antibiotics derive their name from the characteristic configuration
of their molecule, which consists of a fiat aromatic nucleus and a long aliphatic
bridge, shaped like a handle (L. ansa), joining two non-adjacent positions of the
nucleus (Fig. 1) l, 2). The molecules thus formed are very rigid and compact (Fig. 2),
a fact which leads to unique chemical properties and a variety of specific biological
actions. The first compounds isolated some 20 years ago belonged to the group of
rifamycins 3). Since then several other groups have been identified. In the following
article the chemistry, the biosynthesis and the biological activities of the various
ansamycins will be discussed.
Fig. 1. Schematic structure of the ansamycins
Fig. 2 a. Stereomodel of rifamycin SV. The hydrophobic face

1. C h e m i s t r y
Figure 3 shows the chemical structures of the various groups of ansamycins. Two
types of aromatic nucleus can be distinguished: a naphthalenic ring system, as in
the rifamycins, streptovaricins, tolypomycins and naphthomycin and a benzenic
ring system as in geldanamycin and the maytansine group. Except in this last group,
the nuclei are substituted in positions 1 and 4, in many cases forming quinone-hydro22
Ansamycins - Chemistry, Biosynthesis and Biological Activity
Fig. 2b. Stereomodel of rifamycin SV. The hydrophilic face

quinone systems or being easily converted into such systems. The ansa bridge is
always an aliphatic chain containing no lactone bonds, and is linked as an amide to
an amino group of the nucleus. This feature is in contrast to the structure of the
macrolide antibiotics, which characteristically contain a large lactone ring.
The chemistry of the various groups of ansamycins is only discussed briefly, as
several excellent reviews have already been published in recent years l' a, 5). These
should be consulted for detailed chemical descriptions and extensive literature
references.
1.1. Rifamycins
The rifamycins were first isolated by Sensi et aL 3) from Nocardia mediterranei as a
complex mixture (Rifamycins A - E ) . Addition of diethylbarbiturate to the fermentation medium led to the sole production of rifamycin B 6) which was obtained in
crystalline form. Its structure has been determined by chemical 7' 8) and X-ray analysis9). The rifamycins might easily have excaped detection altogether, since rifamycin B has no antibacterial activity. However, it is spontaneously oxidized to rifamycin O and hydrolyzed to rifamycin S, a naphthoquinone derivative; reduction yields
the naphthohydroquinone derivative rifamycin SV (Fig. 4). These compounds inhibit
the growth of Gram-positive bacteria at concentration as low as 0.0025/ag/ml.
The rifamycins have some remarkable chemical properties. Rifamycin SV can
be obtained from the quinone rifamycin S by treating the latter with weak reducing
agents such as ascorbic acid. It is fairly stable against oxidation by air and can form
a stable sodium salt. This behaviour can be ascribed to the acylamino group in position 2. Unsubstituted naphthoquinone can only be reduced under much more
drastic conditions, and once in the hydroquinone form it is very sensitive to oxidation.
23
W. Welu'li
C.H3 CH3 CH3
CH3COO~19
29['.-
CH3 CH3
C H 2 C O O ~
J9
OH Off'~'~'CHa
Ha;._~a
NH
O3o
Tolypomycin Y
(823)
Rifamycin B
(756)
CH3
CHs
NH CH3
HaC~.~
u OCOCH3
Naphthomycin
(720)
Streptovaricin B
(812)
H
HsC
CH3 CH30CH3
O ~
s
CH30 OH
f' ~
CH3
C]-I3 0
O
C H S I ~ HN
CH3Of
O
Geldanamycin
(560)
OCtts
Maytansine
(692)
Fig. 3. Structure of various ansamycins. (Numbers in brackets indicate molecular weights. For
all naphthalenic ansamycins the numbering system as proposed by Prelog7, 8) has been used.)
Another peculiar property of the rifamycins is their high lipophilicity. Even the
sodium salt of rifamycin SV is easily soluble in organic solvents such as chloroform.
One striking feature that emerges upon analysis of the tertiary structure is that all
the oxygen functions of the a n s a ring are situated on the same side as the C-1 and
C-8 hydroxyls of the chromophore (Fig. 2). The rifamycin molecule thus has two
faces differing in lipophilicity. The sodium salts of many rifamycin derivatives have
a tendency to form gels (Dr. W. Kump, personal communication). Furthermore,
derivatives with a lipophilic side chain in position 3 at concentrations as low as
24
Ansamycins- Chemistry Biosynthesisand BiologicalActivity
C H 3 C O O ~
3
HOCH2COOH
, r
o. ?. y
~NH__~
2 ~,\
HjC
OCH~COOH
0
Rifamycin B
/ / o
OH I
~ N H
0 ~ ~0
[
[
CH2-CO
_~_ ~ N H
{{
OH
Rifamycin O
RifamycinS
RifamycinSV
Fig. 4. Structural relations between rifamycinsB, O, S and SV
CH3COO~3
OH OH T
0------7~k
H3C
0
Oil
Rifamycin SV
OH i
NH
CHO
OH [
~ ~ N t t
/
OH
3 - Formylrifamycin SV
/----k
~ "CH=N-N N-CH B
OH
k___./
Rifampicin
(MW 823)
Fig. 5. Structure of rifampicin and its synthesisfrom rifamycin SV
50/lg/ml have been found to form viscous solutions and to sediment when centrifuged at high speed (unpublished results). These observations point to the existance
of intermolecular interactions leading to the formation of aggregates and micelles,
a behaviour comparable to that found with detergents. These chemical properties
should be kept in mind, when rifamycins and other ansamycins are used at high
concentrations as inhibitors of biological systems.
Several hundred semisynthetic derivatives have been prepared in an effort to
obtain substances with better biological activities (for references see Ref.S)). Particularly positions 3 and 4 of the naphthoquinone ring system (numbering system as
proposed by Prelog 7' 8) have been extensively substituted, since it has been shown
that structural changes in these two positions do not critically affect the action of
the substance on the target enzyme, the bacterial RNA polymerase (cf. Chapter 3.).
They can, however, influence other parameters such as its ability to penetrate into
cells, its pharmacokinetic properties and resorption, which are all important for
clinical use as an antibiotic. Rifampicin (U.S.: rifampin), which is a widely used
orally active tuberculostatic agent, is a 3-(4-methyl piperazinyl)-iminomethyl derivative of rifamycin SV, synthesized via the 3-formyl derivative (Fig. 5) l~
25
W. Wehrli
CH3 C,H3 C,H3
H O ~ , 9
Ctl3 CH3 CHa
CH3COO~
~'~ '~o o
o---l--~o o
CH3
CH3
Rifamycin W
Rifamycin G
CH3 CH3 CH3

C H 3 C O O ~
CH3 CH3 CH3

C H a C O O ~
~
OH oH
"c 3
R:r
CH3
CH3
Rifamycin Y
RI
3-Methylthiorifamycin SV SCH3
Halomicin B
R2
OH
I~
CH3
@~--~OH
Fig. 6. Structure of some naturally occurring rifamycins and halomicin B
Many chemically different rifamycins have been isolated from the fermentation
broth of naturally occurring strains of Actinomycetes, or from selected mutants s' 10.
The most interesting ones are rifamycin W t2, D), G14) and yl5, t6) Fig. 6). The structure of rifamycin W bears a remarkable resemblance to the streptovaficins, because
it lacks the ketal linkage between the a n s a chain and the chromophore and has an
extra carbon on C-28. The isolation of rifamycin W contributed greatly towards
the understanding of the biosynthesis of the ansamycins (cf. Chapter 2.). Rifamycin
G differs from rifamycin S in having no double bond C-16 - C-17 and no C-1.
Rifamycin Y is similar to rifamycin B, but has an additional hydroxyl group at C-20
and a keto instead of a hydroxyl group at C-21. These three compounds have proved
very valuable in studies of the relations between the chemical structure and the
biological activity: they are totally inactive in inhibiting bacterial growth as well as
the enzymatic activity of bacterial RNA polymerase (cf. Chap. 3.). Recently, structural studies of the group of halomicins isolated from Micromonospora halophytica 17)
have shown that halomicin B ~8) is closely related to rifamycin B, having a pyrrolidine
26
Ansamycins - Chemistry, Biosynthesisand BiologicalActivity

group on C-4 (Fig. 6). Furthermore, 3-methylthiorifamycin SV has been isolated
from a species of Micromonospora 19). Thus, Nocardia is not the only producer of
rifamycins.
1.2. Streptovaricins
The streptovaricins, produced by Streptomyces spectabilis, are a complex mixture
of closely related substances. Figure 7 shows the formulas of streptovaricins A - G
and J. A summary account of the structural studies undertaken so far has been
published by Rinehart 4). The streptovaricins are chemically related to the rifamycins, but there are a number o f important structural differences between the two
groups, e.g. :
a) the ansa chain is linked to the chromophore by a C - C double bond and not
via oxygen;
b) the configuration of the conjugated double bonds in the ansa ring is different;
c) the B-ring of the naphthalene chromophore is not a benzene ring, but part
of a quinone methide system;
d) the hydroxyl group at C-4 is acetylated;"
e) C-6 and C-11 are linked via a methylenedioxi-bridge.
CHaOOC
R 3 0
CHa CH 3
~
~
A
.o\ A.cV?
r
II
H~C'~,,
-CH3 -
O~-..I ~
,o. o. y
"c.3
r y--f--cH
C
D
E
G
R2
R3
R4
OH
H
OH
OH
COCH3
COCH3
H
H
H
OH
OH
=O
H
H
H
OH
OH
OH
OH
OH
H
OCOCH3
Streptovaricins A-E, G and J.
J
"O._._9 OCOCH~
R1
H
H
OH
OH
OH
CH 3 CHa
HO
CH3
Streptovaricin F
Fig. 7. Structure of various streptovaricins

27
W. Wehrli
On the other hand, the configuration of the 8 chiral centres C-20 to C-27 and
of the ansa system is identical to that found in the rifamycins and in tolypomycin Y.
The streptovaricins differ from each other mainly in the different extent of
oxidation of the ansa bridge 2~ Very recently, protostreptovaricins I - V 21) and
damavaricin C and D 22) have been isolated and structurally analyzed (Fig. 8). It has
been postulated that these compounds are biosynthetic precursors of the streptovaricins 21) (cf. Chap. 2.). Damavaricin C can also be generated from streptovaricin C
by oxidative hydrolysis 23).
HO
R2 CH3 CH3
I
II
Ill
IV
V
R3
R1
R2
R3
H
CH3
H
CH3
CH3
CH3
CH3
CH3
H
H
OH
OH
Proto streptovaricins I - V
H3C' ' v
~"~-0 0
Co3OOC
H3C2 '
CH3 CH3
%0 o
Damavaricin
Fig. 8. Structure of the protostreptovaricins and damavaricins
1.3. Tolypomycin Y
Tolypomycin Y has been isolated from Streptomyces tolypophorus 24-26) together
with rifamycin B and O. Its structure has been elucidated chemically and by X-ray
analysis27) and closely resembles that of the rifamycins, especially rifamycin S
(Fig. 3). In position 4 the chromophore contains a tolyposamin residue which upon
mild acid hydrolysis can be split off yielding the 1,4-naphthoquinone tolypomycinone 24). In the ansa ring, C-19, C-20 and C-31 form a cyclopropane ring and at
C-18 a keto group is found. It should be noted that tolypomycin Y bears no particular resemblance to rifamycin Y; the latter contains a keto group at C-21 and is
biologically inactive, whereas tolypomycin Y has the normal C-21 hydroxyl function and is biologically active.
28
Ansamycins - Chemistry Biosynthesisand BiologicalActivity

1.4. Naphthomycin
Naphthomycin has been isolated from a Streptomyces species 2a). Its structure has
been elucidated and shown to belong to the class of naphthalenic ansamycins
(Fig. 3) 29). In contrast to these compounds, however, the skeleton of the ansa
chain contains six additional C-atoms. Very recent structural studies of the chromophore have shown that naphthomycin possesses a hydroxyl group in position 6 and
is unsubstituted in position 8 (W. Keller-Schierlein, personal communication). It
thus resembles the streptovaricin precursor protostreptovaricin I (Fig. 8). A further
characteristic of naphthomycin is that it contains a halogen in the chromophore.
As a result of these structural properties, its biological effects differ from those of
the other naphthalenic ansamycins (of. Chap. 3.).
1.5. Geldanamycin
In contrast to the foregoing ansamycins which all contain a naphthalenic ring system,
the chromophore ofgeldanamycin, an antibiotic isolated from Streptomyces hygroscopicus 30), is a benzoquinone derivative (Fig. 3) 31). The ansa ring between C-1 and
C-11 resembles the part of the other ansamycins between C-15 and C-25. C-12 to
C-15 seem to correspond to C-5 to C-8 of the naphthalene skeleton. At C-21 a carbamoyl residue is found. Considering all these marked structural disparities, it is not
surprising that the biological activity of geldanamycin differs from that of the naphthalenic ansamycins (cf. Chap. 3.) However, an analogous biosynthesis (Chap. 2.)
clearly assigns it to the class of ansamycins.
1.6. Maytansine and Related Compounds

Maytansine and other maytansinoids 32-37) are the only ansamycins so far found to
occur in plants (Celastraceae, Rhamnaceae, see Table 1). Since wood and bark of the
plants in question only contain about 0.7 ppm, 10 tons had to be processed to isolated some 6 g of maytansine. The chemical structure (Fig. 9) resembles that of
geldanamyein, having a benzenic chromophore and the same number of C-atoms in
the skeleton of the ansa ring. However, C-7 and C-9 are linked forming a carbinolamide, and maytansine and other members of the group contain a fairly large substituent at C-3. In addition, an epoxy group is found in a position adjacent to that
in tolypomycins and a chlorine in the same position as in naphthomycin. X-ray
analysis of (3-bromopropyl)-maytansine revealed that the 19-membered ansa ring
has two roughly parallel sides, C-1 to C-6 and C-IO to C-15, which are linked on
one side by the chromophore and on the other side by the six-membered carbinolamide ring 32' 37). Thus a rather fiat molecule is formed with two faces of different
character: that with the C-3 ester and C-9 hydroxyl group is hydrophilic, the other
predominantly hydrophobic, a situation analogous to that found in the rifamycins.
A large number of maytansinoids have been isolated and chemically characterized, and interesting structure-activity relationships have been deduced, suggesting
29
W, Wehrli
that the C-3 ester and the C-9 hydroxyl group are essential for biological activity
(Chap. 3.).
HsCO OH
('H3
OR1
,~c- \
R2~-~NX'cH
" ~ "Cl
OCH3
Rl
Maytansine
R2
- CH - N - C - C H
II
CH3 CH30
Maytanbutine
CH- N -C-CH-CH 3
O
Maytanvaline
-C
Maytanacine
-C
II
CH3
CH- N -C-CH2-CH-CH 3
H t
CH3 CH30
ti
CH3 CH30
CH3
CH3
II
Maytansinol
-H
Colubrinol
- C - C H - N - C - CH - C H 3
II I
II
O CH3 CHa O
OH
CH3
H3CO OH
C
L
OCH3
Maysine
Fig. 9 Structure of maytansine and related compounds
30
CHa
~
]j~NH
OCH3
Maysenine

1.7. Streptolydigin and Tirandamycin
Neither streptolydigin 38) nor tirandamycin 39) belong to the class of ansamycins,
but they have some biological activities resembling those of the ansamycins
(cf. Chap. 3.), and also certain notable structural analogies (Fig. I0): both streptolydigin and tirandamycin contain a great part of the a n s a ring system found in the
ansamycins. Thus it could be postulated that the similarity of the a n s a rings is the
reason for the analogies found in their biological action.
CH3 CH3 CH3
~
HO..~,~
H
~H--'CONHCH3
CH3
Streptolydigin
Tirandamycin
Fig. 10. Structure of streptolydigin and tirandamycin
2. Biosynthesis
Although all ansamycins consist of an aromatic nucleus spanned by an aliphatic bridge,
the chemical structures of these two parts vary considerably. Moreover, ansamycins
are isolated from a variety of actinomycetes and even from plants (Table 1). On the
other hand, the similarities between the various members of the group are striking.
An analogous configuration occurs, for instance, at all 8 asymmetrical C-atoms
(C-20-C-27) of the a n s a ring in rifamycins, streptovaricins and tolypomycins l). It
could therefore be postulated that the ansamycins have a common route of biosynthesis. Certain similarities to macrolide antibiotics such as erythromycin suggested
that the ansamycins might be synthesized in a similar manner. Woodward4~ proposed
that macrolides are formed from acetic acid and propionic acid residues in an analogous way as fatty acids are synthesized from acetic acid. Birch 40 put forward the
hypothesis that the methyl groups might be introduced into an intermediate polyketide chain by transmethylation through compounds such as methionine or choline.
31
W. Wehrli
Table 1. Origin and biological activities of ansamycins

Compound
Origin
Inhibition of
bacterial RNA
polymerase
Rifamycins
Nocardia mediterranea
Micromonospora halophytica etc.
Streptomyces tolypophorus
Streptovaricins
Tolypomyein
Streptomyces spectabitis
Streptomyces tolypophorus
+
+
Naphthomycin
Streptomyces collinus
Geldanamycin
Streptomyces hygroscopicus
Maytansines
Maytenus serrata
I
Maytenus buchananii (Celastraceae)
Putterlickia verrucosa
Colubrina texensis
(Rhamnaceae)
Biological
activities
Antibacterial
(antifungal,
antivixal, antiturnout)
Antibacterial
Antibacterial,
antifungal
Antibacterial,
antiprotozoal
Antimitotic,
antileukaemic,
antitumour
Direction of biosynthesis
Precursors:
e'X---- methylmalonat e
=
malonate
v acetate
o methionine
Fig. 11. Scheme of the biosynthesis of rifamycin S
Investigations o f the biosynthesis o f the rifamycins, streptovaricins and geldanamycin

proved that the ansa chain of the ansamycins is synthesized as proposed by Woodward. The isolation o f the precursors rifamycin W, damavaricin C and D and the
protostreptovaricins, together with the fact that rifamycin B and tolypomycin Y
are cosynthesized by Streptomyces tolypophorus 26) make it probable that the rifamycins, streptovaricins and tolypomycin Y have a common progenitor.
32

2.1. Rifamycins
The biosynthesis of the rifamycins was studied by using either 14C. or 3H-labelled
precursors and subjecting the labelled product to extensive chemical degradation
and analysis, or using 13C-enriched precursors and analyzing the product by carbon.13
magnetic resonance spectroscopy42-44). These experiments have shown (Fig. 11)
that to a CTN piece of as yet uncertain origin 4s) eight propionate and two acetate
units are added to form the chromophore and the ansa ring of rifamycin S, whereby
the incorporation of the propionate occurs via methylmalonate and the acetate
incorporation via malonate. Only the methyl of the -OCH 3 group at C-27 stems
from methionine, and it is introduced after completion of the ansa ring. The ansa
chain grows clockwise and ends at the C-15 carbon atom (Fig. 11). The first precursor product is thought to be modified by cleavage of the methyl group on C-28
and by oxidation of the ansa chain between C-12 and C-29 to yield a ketal linkage.
That this does in fact occur has been neatly demonstrated by the isolation of rifamycin W 12) (Fig. 6), in which C-12 and C-29 are directly linked by a C---C double bond,
and a hydroxymethyl group is still found at C-28. Rifamycin W can be transformed
by the parent Nocardia strain to rifamycin B, and it is thus thought to be a normal
intermediate in the formation of the other rifamycins.
Rifamycin S has been shown to be further processed to rifamycin B46' 47) or
rifamycin G t4). Rifamycin B and rifamyein S can be oxidized to yield rifamycin Y
and rifamycin YS respectively48). Figure 12 summarizes the route of biosynthesis
of the rifamycins based on these findings.
2 acetate + 8 propionate + " x ~
rifam, 'tin
rifam, 'cin
~ rifamycin L
q> rifamycin G
I> rifamycin YS
rifam cln
D rifamycin Y
Fig. 12. Proposed route of the biosynthesis of the rifamycins44)
2.2. Streptovarieins
Using carbon-13 magnetic resonance spectroscopy, Rinehart and his collaborators
have shown 49) that the biosynthesis of the streptovaricins is very similar to that of
the rifamycins. Streptovaricin D is synthesized from a CTN unit of unknown origin
to which 8 propionic acid residues and two acetic acid residues are attached, whereby the direction of growth is the same as that of the rifamycins. In contrast to the
33
W. Wehrli
2 acetate + 8 propionate + ~
protostreptovaricins I
damavaricin D
protostreptovaricin II
streptovaricin
streptovariein
A <1~
protostreptovaricin IV
-1>streptovaricin
streptovaficin B
damavaricin
C
t2
streptovaricinE, 13, J
Fig. 13. Proposed routes of biosynthesis of the streptovaricins21)

rifamycins, the streptovaricins retain an intact double bond between the C-12 and
C-29 and the methyl group on C-28 is not split off. On the other hand, the hydroxyl
group in position 6 on the chromophore is methylated, methionine being the methyl
donor, then processed in such a way that C-6 and C-1 1 are linked by a methylenedioxi-bridge. Besides, the methyl group at C-3 also stems from methionine 22).
As possible biogenetic precursors of the streptovaricins, damavaricin C and D
and the protostreptovaricins I - V have been isolated and characterized (Fig. 8) 21' 22).
Streptovaricin C and D are the precursors of the other streptovaricins22). Figure 13
summarizes the various supposed routes of biosynthesis of the streptovaricins.
2.3. Geldanamycinand Maytansine

Studies using ~4C-labelled precursors and C-13 carbon magnetic resonance have
suggested that the biosynthesis of the benzenic ansamycin geldanamycin follows
essentially the same pathway as that of the rifamycins and streptovaricins 5~ Geldanamycin is composed of 3 acetate and four propionate units which are attached to
a C7 N unit in the same direction of growth as is found in rifamycins and streptovaricins. The incorporation of three acetate units into the a n s a chain, as opposed to
two in the naphthalenic ansamycins, excludes the existance of a common precursor.
Nothing is known about the biosynthesis of the maytansine group, the only
ansamycins so far identified as being o f plant origin. Their similarity to the bacterial metabolite geldanamycin has led to the hypothesis that despite their occurrence
in plants, microorganisms might be involved in their production.
3. Biological A c t i o n s
The ansamycins have a very broad spectrum of biological effects which are of great
significance for both scientific and practical reasons. One member of the rifamycins,
34

rifampicin, has proved to be an excellent, orally active antibiotic and is now in widespread clinical use, especially as an antituberculous agent but also in the treatment
of various other infective diseases.
From the biochemical point of view, the unique mode o f action of some of the
ansamycins, in particular of rifampicin, has aroused much interest. Rifampicin and
several other ansamycins have been shown to inhibit bacterial transcription very
specifically and at extremely low concentrations by interacting exclusively with
DNA-dependent RNA polymerase. This unique action has spurred many investigations on the effects of ansamycins in a variety of viral and eukaryotic systems. More
recently maytansine and related compounds have been found to be very potent antimitotic agents and to have interesting antitumour activities.
The published literature on the biological and medical actions of ansamycins is
too vast to be dealt with at length in this article. Several reviews have appeared, however, dealing with the rifamycinssl-s3)
3.1. The Various Modes of Action
Table 1 indicates the main biological activities of ansamycins. In analysing their great
variety it seems useful to group them according to the drug concentration needed to
elicit an effect in vitro on isolated enzyme systems or on intact bacterial or eukaryotic cells. The reason for this is that effects evoked by low drug concentrations point
to a specific interaction with a def'med receptor molecule, whereas at high drug levels
"effects" may be artifacts or due to unspecific actions, as has unfortunately been the
case with many experiments done with ansamycins.
3.1.1. Effects Produced by Drug Concentrations Below 1 tag/ml ( < 10 -6 M)
I. Effect on D N A transcription in bacteria:
The rifamycins, streptovaricins and tolypomycins are very effective antibacterial

agents. They all inhibit the synthesis of RNA by inactivating the DNA-dependent
RNA polymerase. This effect occurs at low concentrations (0.01 tag/ml, 10 -a M)
and is highly specific. It is the most thoroughly investigated and clearly defined
biological action of the ansamycins.
11. Effect on mitosis:
Maytansine and related compounds inhibit cell division in sea urchin eggs at
concentrations of 0.04/ag/ml (6 x 10 -8 M), possibly by interfering with the polymerization of tubulin. This effect bears some resemblance to the action of the vinca
alkaloids such as vincristine. Maytansine inhibits the growth of KB ceils at levels of
10- s/.tg/ml.
3.1.2. Effects Produced by Drug Concentrations Over 1/ag/ml ( > 10 -6 M)
L Actions on eukaryotes:
Clear evidence exists to prove that ansamycins such as rifampicin have no effect
on eukaryotic RNA polymerases, be they of nuclear, mitochondrial or chloroplastic
35
W. Wehrli
origin. Certain reports claiming inhibition of mitochondrial or chloroplastic RNA
polymerase are doubtful because of the experimental conditions used or the high
drug concentration needed as compared to that required for inhibition of the bacterial enzyme. Rifamycins with lipophilic side chains and some derivatives of streptovaricin and geldanamycin have been found to inhibit indiscriminately a large number
of both DNA and RNA polymerases of bacterial, eukaryotic and viral origin. The
100-10'000-fold higher drug concentration needed for inhibition, as well as the
lack of enzyme specificity, definitely distinguishes this effect from the inhibition of
bacterial RNA polymerase.
H. Effects on R N A turnout viruses:
As has already been mentioned, some lipophilic rifamycins and some streptovaricins and geldanamycins affect the growth of cells transformed by RNA tumour
viruses or the RNA-dependent DNA polymerase (reverse transcriptase) characteristic
of these viruses. Again, high drug concentrations are needed to produce an effect
and only partial, but never absolute, selectivity of enzyme inhibition has been found.
IlL Effects on DNA viruses and larger infectious agents belonging to the genus o f
Chlamydiae:
Certain ansamycin derivatives, such as rifampicin, inhibit the growth of these
organisms at high drug concentration. The underlying mechanism of action is poorly
unterstood at present, but it does not seem to be related to RNA or DNA synthesis.
3.2. Effects on Bacteria

3.2.1. Interaction with DNA-dependent RNA Polymerase
The potent antibacterial activity of the rifamycins, streptovaricins and tolypomycins
is a consequence of the specific inhibition of DNA-dependent RNA polymerase, the
enzyme responsible for most of the transcription of DNA to RNA. The interaction
between these ansamycins and the enzyme has accordingly been studied in great
detail. Because of its easy availability, rifampicin has been used most often as the
model compound in such studies, but it is reasonable to assume that the data obtained with rifampicin also hold true, at least qualitatively, for the other rifamycins,
the streptovaricins and tolypomycins.
Rifampicin was first shown by Hartmann et al. s4) to have a specific inhibitory
effect on RNA polymerase from E. coli. Later, other active ansamycins were found
and RNA polymerases from a large variety of bacteria other than E. coil proved to be
sensitive to the drug. More recently, an RNA polymerase from E. coli containing only
one subunit and probably involved in the initiation of DNA replication (dna G gene
product) has been shown to be resistant to rifampicin ss). This holds true also for the
various mammalian RNA polymerases. In contrast to non-specific inhibitors of transcription such as actinomycin and mitomycin, rifampicin interacts specifically with
the bacterial enzyme itself. With the aid of 1ac_labelled rifampicin it could be shown
that the drug forms a very stable complex with the enzyme in a molar ratio of
1 : 156, 57). The dissociation constant of this complex is 10 - 9 M at 37 ~ and
36

2.7 x 10 -1~ M at 0 ~ sa' sg). These relatively low constants explain why ansamycins,
and in particular rifampicin, inhibit both the activity of RNA polymerase and the
growth of bacteria at very low drug concentrations. Experiments with rifampicinresistant mutants have shown 6~ 61) that the 15subunit of RNA polymerase contains
the binding site for the drug. It could be demonstrated, however, that binding to the
enzyme precursor a2~ is very weak 62). To form the specific drug-binding site, the
four subunits a2/3/3', as they occur in the core enzyme, are required, a, the subunit
necessary for specific promoter recognition, affects only weakly the ansamycin
binding sites 8).
3.2.2. Mode of Action
Inhibition of RNA polymerase is a direct consequence of the binding of the drug to
the enzyme: on the one hand, enzyme inhibition only occurs, when the ansamycin
is bound, and on the other hand, drug binding never seems to occur without causing
inhibition.
Two aspects of the mode of action must therefore be distinguished:
a) what determines and influences the binding of ansamycins to RNA polymerase
b) which function of RNA polymerase is impaired when ansamycins are bound
to the enzyme.
Recent investigations of the rate of binding of rifampicin to RNA polymerase
at various stages of transcription have shown that nucleic acids bound to the enzyme
strongly decrease the on-rate of the antibiotic to the enzyme sa). Rifampicin binds
500 times slower to a specific RNA polymerase -1"7 DNA complex than to the free
enzyme. The rate of drug binding to a ternary enzyme-DNA-RNA complex, as it
occurs during RNA chain elongation is at least 5 orders of magnitude slower. These
direct kinetic measurements are in line with earlier observations that DNA can
protect RNA polymerase against inactivation by rifampicin 63-6s) and that ansamytins inhibit RNA chain initiation, but not elongation 66). The rate of enzyme inhibition by ansamycins therefore parallels the on-rate of drug binding and can vary
greatly depending on the stage of transcription.
For along time there was no clear picture as to which function of the RNA
polymerase was impaired by ansamycins. Very recently, McClure and collaborators
clarified the problem in a series of very elegant experiments 67), personal communication. Upon analyzing the initiation of RNA synthesis on the PR promoter of phage ),,
McClure found that rifampicin leads to abortive initiation, i.e. to the formation of
the dinucleotide pppApU, and inhibits the formation of the second phosphodiester
bond. A model is proposed in which rifampicin binding to RNA polymerase steritally blocks the translocation of pppApU. The drug does not affect the recognition
of the specific promoter by the enzyme, since the only dinucleotide formed is
pppApU, which corresponds to the first two bases of the gene starting at PR3.2.3. Structure-Activity Relations
The antibacterial activity of the ansamycins varies to a large extent. In most cases,
antibacterial activity parallels the inhibition of the bacterial RNA polymerase and
37
w. Wehrli
can therefore be predicted to some extent on the basis of structure-activity studies
with the enzyme. The availability of many natural and semisynthetic derivatives,
especially in the rifamycin and streptovaricin groups, has made it possible to get an
idea of the structural parameters necessary to inhibit RNA polymerase. For the
rifamycins, some Of the main features are the following6s' 69).
1. Free hydroxyl or keto groups at C l and C8;
2. unbroken ansa-bridge;
3. free hydroxyl groups at C21 and C23.
Analysis by X-ray69) and NMR spectroscopy7~ shows that the rifamycin molecule is very rigid (Fig. 2). The four oxygen functions at Cl, Cs, C21 and C2a all lie
on the same side of the molecule. Being required for enzyme inhibition it is reasonable to assume that they are involved in the binding of the drug to the enzyme.
However, kinetic studies of the interaction of rifampicin with RNA polymerase in
various solvents indicate that the bonds forming the drug-enzyme complex are mostly of a lipophilic nature s8). Large parts of the ansa ring thus seem to participate in
the binding to the enzyme as well as the chromophore which possibly interacts with
an aromatic amino acid, since rifampicin shows a characteristic bathochromic shift
when bound t o the enzyme. The nature of the interactions between the drug and
the enzyme could therefore conceivably be represented by the model shown in
Fig. 14, in which the enzyme closes on the drug molecule from three sides. In such
a model, positions 3 and 4 on the chromophore do not take part in the binding.
This would be consistent with the finding that chemical substitutions at C-3 and
C-4 of the molecule in many cases have little effect on its interaction with the enzyme68, 71). Effects on eukaryotic and viral enzymes observed after the introduction
of substituents in these positions are most probably not due to specific binding such
as is found with the bacterial enzyme.
Although many facts are known about the structure.activity relations ofansamycins, some problems remain unsolved. Rifamycin W, for instance, (Fig. 6) does not
rifc
Chromol
Fig. 14. Modelof the interaction between rffampicin and RNA polymerase
38

seem to inhibit RNA polymerase ll), although the structural requirements mentioned
above are fulfilled. On the other hand, damavaricin C, which is assumed to be a precursor of streptovaricin C and is chemically very similar to rifamycin W, is a powerful inhibitor of the enzyme 23). Moreover, among the streptovaricins, streptovaricin J
(acetate at C-21) and streptovaricin E (keto group at C.21) still show some activity,
whereas the corresponding rifamycin-C-21-acetate and rifamycin Y are inactive 2~
The structure-activity relations of the rifamycins and the streptovaricins therefore
do not seem to be identical.
Another puzzle is posed by naphthomycin. The fact that it does not inhibit RNA
polymerase can be explained by the absence of a hydroxyl group at C-8. However, it
shows antibacterial activity against gram-positive strains that is counteracted by SHcontaining substances such as cystein2s). Naphthomycin is therefore an example of
an ansamycin with antibacterial activity not due to inhibition of RNA polymerase.
The structure of the ansamycins determines not only their activity on RNA
polymerase, but also other important characteristics such as their ability to penetrate into bacteria and their pharmacokineties and absorption in the host. To cite
just a few examples: rifamycin B, containing a free carboxylic acid group, has no
antibacterial activity, although it inhibits RNA polymerase as strongly as rifampicin.
Damavaricin C behaves similarly to rifamycin B, whereas its 6-methyl ether inhibits
RNA polymerase to a lesser extent, but has good antibacterial activity2a). Rifampicin owes its widespread clinical use to the fact that, in contrast to most other
rifamycin derivatives, it is well absorbed when given orally.
3.2.4. Nature of Ansamycin Resistance in Mutants

Mutants resistant to rifamycins have been isolated from a variety of micro-organisms.
The mutation rate ofE. coli is in the region of 10 -a, and the conversion is apparently
due to a one-step mutation. The mutants analyzed so far map at a single position
(79 minutes) on the E. coil chromosome 72). RNA polymerase has been prepared
from rifamycin-resistant mutants ofE. coli and S. aureus. In both cases it was found
that the enzyme differed from the corresponding enzyme of the sensitive strain in
that it is no longer inhibited by rifampicin and, in addition, could no longer bind the
drugS6, 73, 74). Resistance to rifampicin was therefore caused by an alteration in the
structure of the enzyme, probably by the replacement of one single amino acid. In a
number of resistant mutants, the site of the mutation was located in the fl subunit 6~ 61).
Resistance to rifampicin is not an all-or.nothing phenomenon. Mutants can be
selected that are resistant to various concentrations of the drug. As the bacteria
become more resistant to rifampicin, the sensitivity of the corresponding RNA polymerase decreases, and the enzyme-antibiotic complex becomes less stable 7s). Hence,
the degree to which the enzyme and the antibiotic fit each other varies widely, indicating that there are many possibilities for the location and the nature of the amino
acid substitution in the enzyme molecule. These substitutions would be expected to
occur mainly in the ~ subunit, but mutations in other subunits such as ~' or t~ cannot
be excluded, since, as discussed above, the core enzyme 0~2/~' has to be present for
the formation of the specific drug-binding site.
39
w. Wehrli
The possible occurrence of ansamycin resistant mutants in which at the same
time the correct functioning of the RNA polymerase has been affected poses an
interesting problem. Recently such mutants have been found in Lactobacillus casei
which were resistant to rifampicin and in the same mutational event had developed
auxotrophy for glutamine76). A rifampicin resistant mutant ofE. coli W has been
found to contain an alteration in some parameters regulating the arglnine biosynthesis 77). Such pleiotropic effects could be of great help in elucidating the different
modes of the regulation of gene expression.
As would be expected from their similar mechanisms of action, resistance to
streptovaricins and tolypomycins develops in a way analogous to that found with
the rifamycins, and cross-resistance is observed between all three groups of antibiotics78, 79)
So far no enzyme has been found that can inactivate ansamycins either by cleavage or by chemical modification.
3.2.5. Other Effects on Bacteria
As has already been mentioned, naphthomyein inhibits grampositive bacteria,
although it does not inhibit RNA polymerase. An antagonism between naphthomycin and vitamin K has been observed2a), but the underlying mode of action is not
known.
The two structurally related antibiotics streptolydigin and tirandamycin should
be mentioned at this point. They do not belong to the ansamycins, but show some
striking similarities in their chemical structure (Fig. 10). Surprisingly enough, these
two compounds also inhibit RNA polymerase by binding to the 13subunit, although
higher drug concentrations are required and their mode of action differs from that
of the ansamycins6~ so-a2). Genetic studies ofE. eoli have shown that the loci for
rifampicin and streptolydigin resistance map very closely together sa). It could be
postulated that the binding sites for streptolydigin and rifampicin partially overlap.
3.3. Effects on Eukaryotes

The rifamycins, streptovaricins and tolypomycins which inhibit bacterial RNA polymerase, do not in general affect eukaryotic RNA polymerases, whether they are of
nuclear, mitochondrial or chloroplastic origin s2). The antibacterial action of these
ansamycins resides in the recognition of a specific binding site on the bacterial
enzyme that is absent in eukaryotic enzymes. Certain ansamycins such as lipophilic
rifamycin derivatives at high concentrations show some activity on a large variety of
mammalian DNA and RNA polymerases as well as on rifampicin-resistant bacterial
RNA polymerase s2, 84-87). Even enzymes other than polymerases, such as glutamateoxaloacetate-transaminase (GOT), glutamate-pyruvate-transaminase (GPT) and alkaline-phosphatases, and polyphenylalanine synthesis in a cell-free system are inhibited
to some extent sT' ss). This inhibition is due to non-specific binding of the drug to
40

these enzymes, whereby the number of drug molecules bound depends on their concentration and can amount to several hundreds per molecule o f the enzyme 88).
Studies of sedimentation and viscosity show that lipophilic rifamycins form large
aggregates at the same concentrations as those leading to enzyme inhibition (unpublished observation). These data clearly demonstrate that the inhibitory action
on these enzymes cannot be related to the very specific interaction of some ansamycins with bacterial RNA polymerase, which occurs at 1000 to I0'000 times lower
concentrations and in a very well defined stoichiometric ratio of 1 : 1.
Nevertheless, under certain conditions some ansamycins seem to affect the
growth of eukaryotic cells. Medoff and collaborators89' 9o), for instance, have
reported that rifampicin together with amphotericin B inhibited the growth of certain yeasts such as S. cerevisiae and C. albicans, whereas neither antibiotic alone had
any effect at the concentrations tested. This observation has been confirmed by
other authors with a variety of different fungi 91-94) and even with mouse L-cells9s).
The underlying mechanism of action is rather puzzling. On one hand, there seems to
be some indication that RNA synthesis is inhibited, which has led to the conclusion
that amphotericin B facilitated the permeation of rifampicin through the cell membrane, allowing rifampicin to act on RNA synthesis much as it does in bacteria 89).
On the other hand, careful studies have shown that neither the various nuclear 96)
nor mitochondria197) RNA polymerases of yeast are inhibited, even by very high
concentrations of rifampicin.
The results obtained in experiments with a fragile Saccharomyces cerevisiae
mutant are even more puzzling; in contrast to the wild type it was found to be inhibited by rifampicin alone and even seems to contain an RNA polymerase sensitive to
the drug, whereas the analogous enzyme from the wild type is rifampicin-resistant 98).
Similar results have been obtained with vesicular stomatitis virus, an RNA virus conraining an endogenous RNA transcriptase. Here again, both the growth of the mutant
strain and its enzyme are drug sensitive, but the wild type is drug resistant 99). Further
studies are needed to clarify the situation, and in this respect determinations of the
rate of mutation to drug resistance might be of great value.
Very interesting effects on eukaryotic cells result after treatment with ansamycins containing a benzene chromophore, especially with compounds of the maytansine group which are the only ansamycins found so far to occur in plants.
Maytansine and some of its congeners affect the growth of KB cells at concentrations as low as 10 -4 to I0 -s/lg/ml a2). They irreversibly inhibit cell division in
the eggs of sea urchins and clams, causing the disappearance of the mitotic apparatus.
They inhibit the polymerization of tubulin in vitro 100) and thus act similarly to the
vinca alkaloids such as vincristine. Bacterial RNA polymerase is not affected up to a
concentration of 60/~g/ml 1~ Despite its highly toxic effects on cells in vitro, maytansine and other maytansinoids are highly potent antitumour drugs with a relatively
low toxicity in rive. The reason for their specific action remains unclear, but differences in the cell surface affecting drug attachment and permeation might be of
importance. Structure-activity studies have revealed the crucial role of a free hydroxy
group in position 9 and an ester group in position 335, 36) (see Fig. 9). Maytansine
and other maytansinoids esterified at C-3 have a potent antileukaemic effect in the
~tg/kg dose range and are very cytotoxic, whereas compounds without an ester such
41
w. Wehtli
as maytansinol, maysine, maysenine and also geldanamycin lack an antitumour
effect and have a cytotoxicity 3 - 5 orders of magnitude lower. Conversion of the
free C-9 hydroxyl group to the C-9 ethyl ether yields an inactive compound. As to
the mechanism of action, it has been postulated that after specific binding to the
target protein(s), maytansine acts as an alkylating agent of free thio- or amino-groups,
preferably through C-9.
3.4. Effects on RNA Tumour Viruses

Oncogenic RNA viruses contain an RNA-dependent DNA polymerase (reverse transcriptase) that is characteristic of this group of viruses and seems to be of importance
for viral proliferation and the transformation of its host cell 1~ lO3). Since RNA
tumour viruses might be implicated in some human cancers, inhibitors of reverse
transcriptase could be very valuable research tools in defining the exact function of
this enzyme in viral carcinogenesis and they might even be of clinical value. The early
finding that certain lipophilic rifamycin derivatives inhibit reverse transcriptase and
have some effects on transformed cells 1~ 10s) led to great efforts both to synthesize
new ansamycin derivatives and to analyse them in a variety of biological systems s' s2,106)
A large number of rifamycin, streptovaricin and geldanamycin derivatives have been
synthesized, in most cases through substitution of the chromophore. Many of them
inhibit reverse transcriptase to some extent, but none o f the substances so far tested
acts in an absolutely selective way and at concentrations comparable to those that
inhibit bacterial RNA polymerase. In fact, as has already been discussed in the preceeding section, many other nucleic acid polymerases of bacterial, eukaryotic and
viral origin, as well as other enzymes such as phosphatases, are also inhibited to a
similar extent by these derivatives. For all these enzymes, including reverse transcriptase, the drug concentrations required to bring about this effect are relatively
high ( > 1/ag[ml). As in the case of eukaryotic polymerases there is no clearly defined
stoichiometry of binding to reverse transcriptase independent of the drug concentration.
It can therefore be concluded that inhibition of reverse transcriptase caused by
ansamycins with a modified chromophore is not due to a strong and defined interaction between the drug and enzyme, but rather results from the weak binding of a
varying number of aggregated drug molecules to a relatively undefined protein region
occurring in many enzymes. There is little chance that further chemical modification
of the ansamycin chromophore will lead to compounds with a selective activity on
reverse transcriptase at low concentrations.
3.5. Effects on DNA Viruses and Larger Infectious Agents such as Chlamydiae
Most studies about the effects of ansamycins on DNA viruses have been made with
vaccinia virus sl). It has been found that some derivatives, such as rifampicin, inhibit
the growth of this virus. There is no doubt, however, that this inhibition is not due
to a block in RNA synthesis, as was found in bacteria, but apparently the assembly
42

of immature virus particles is affected 1~176 As in the case of the RNA viruses,
very large concentrations of antibiotic are needed to inhibit the virus growth. Thus,
clinical application is not possible.
The infectious elementary bodies of trachoma agent belong to the Chlamydiae
which are parasites of mammalian cells and are considered unusually small bacterial
cells. As in the ease of vaccinia virus, only certain ansamycins, e.g. rifampicin at very
high concentrations, affect the growth of trachoma agent. The mechanism of action
is not known, but again RNA synthesis is not involved.
4. C o n c l u s i o n s and S u m m a r y
The ansamycins are a remarkable group of natural compounds varying widely in both
their chemical structure and their biological activities. They have mostly been isolated
from prokaryotic microorganisms, but one group, the maytansines, occurs in plants.
Their chemical structure consists of two parts, a chromophore and a long aliphatic bridge spa,ning it. The molecules thus formed are very compact and rigid.
As a consequence, intramolecular and intermolecular interactions lead to unexpected
chemical properties. In particular, those derivatives with lipophilic side chains tend
to aggregate and behave like detergents even in dilute solutions. This property should
be taken into account, when ansamycins are used at high concentrations in biological
systems. The ansamysins do not contain lactone bonds in their ansa ring, which sets
them clearly apart from the macrolide antibiotics.
Studies of the biosynthesis of the substances have shown that starting from a
nucleus o f unknown origin a varying number of acetate and propionate units can be
linked to yield ansamycins differing in both the structure of the chromophore and
the ansa chain. Continued research will very probably disclose new types of ansamycins with novel biological actions. One interesting problem in this context is to
determine what modes of regulation exist to direct the biosynthesis of common
building blocks into specific compounds, and whether it might even be possible to
influence and modify this regulation in such a way that new biosynthetic pathways
yield novel compounds.
Ansamycins have been shown to cause a large variety of biological effects on
bacteria, eukaryotes and viruses. Two of these are very powerful and selective. One
is the specific inhibition of bacterial RNA synthesis by rifamycins, streptovaricins
and tolypomycins. Detailed investigations have proved that DNA-dependent RNA
polymerase, the enzyme responsible for DNA transcription, forms a very stable 1 : 1
complex with these ansamycins and as a consequence, is inactivated. Eukaryotic and
viral enzymes do not interact with the drug in this selective manner. The binding of
ansamycins to bacterial RNA polymerase is a good example of a specific drug-receptor interaction; a chemically complex molecule with a rigid shape is linked by
physical bonds to a complementary site of a macromolecule. This results in a tight
complex and, as a consequence, in a dramatic change of the functional properties
9of the macromolecule.
More recently, the maytansines have been found to exert a very potent antimitotic action on eukaryotic cells and to show interesting antitumour activity. The
43
w. Wehrli
drug concentrations necessary to cause these effects are very low which suggests
that the mode of action although not yet known in all its details is selective.
Besides these two specific actions, many other biological effects caused by
ansamycins have been reported. Most of them are unspecific and are only observed
at high drug concentrations, at which the derivatives used have the detergent-like
properties mentioned above. To this category belongs the inhibition of the various
mammalian and viral nucleic acid polymerases, including reverse transcriptase. It
must be stressed very strongly that an effect on a mammalian enzyme at drug concentrations of 5-200/ag/ml cannot be interpreted in the same way as the inhibition
of bacterial RNA polymerase at 10 -2/ag/ml.
Finally, there remain some effects of ansamycins of which the significance and
the biochemical targets are as yet unknown; these include the combined action of
rifamycin and amphotericin B on fungal ceils, and the antibacterial and antifungal
activity of naphthomycin and geldanamycin.
Acknowledgements. I wish to thank Dres. W. Keller-Schierlein,W. Kump and W. R. McClttre for
providiIa~unpublished results and Dres. O. Ghisalba, K. Hauser, J. Handschin, A. H. Kirkwood
and J. Niiesch for their critical review of the manuscript.
44
Ansamycins - Chemistry Biosynthesis and Biological Activity
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Gallo, G.G., Martinelli, E., Pagani, V., Sensi, P.: The confirmation of rifamycin S in
solution by IH NMR spectroscopy. Tetrahedron 30, 3093 (1974)
Dampier, M.F., Whiflock, H.W.: Electronegative gloups at C-3 of rifamycin S enhance its
activity toward DNA-dependent RNA polymerase. J. Amer. Chem. Soc. 97, 6254 (1975)
Taylor, A.L., Trotter, C.D.: Linkage map of E. cell strain K-12 Bact. Rev. 36, 504 (1972)
Wehrli, W., Kniisel, F. Staehelin, M.: Action of rifamycin on RNA polymerase from
sensitive and resistant bacteria. Biochim. Biophys. Res. Commun. 32, 284 (1968)
47
W. Wehrli
74) White, R.J., Lancini, G.C.: Uptake and binding of aH-rifampicin by E. coil and Staph.
aureus. Biochim. Biophys. Acta 240, 429 (1971)
75) Zimmcrmann, W., Wehrli, W.: Rifampicin resistance in E. coli: comparison of
microbiological and enzymatic properties. Experientia 33, 132 (1976)
76) Morishita, T., Yuta, T.: Altered nutritionalrequirements associated with mutations
affecting the stracture of RNA polymerase in Laetobaeillus casei. J. Bact. 125,416 (1976)
77) Wozny, M.E., Carnevale, H.N., Jones, E.E.: Alteration of regulation of arginine biosynthesis
in F,. coil W by mutation to rifampicin resistance. Biochim. Biophys. Acta 383, 106 (1975)
78) Nitta, K., Mizuno, S. Yamazaki, H., Umezawa, H.: Streptovaricin and rffampicin resistance
of RNA polymerase in a resistant clone ofE. r B.J. Antibiotics 21,521 (1968)
79) Kondo, M., Oishi, T., Tsuchiya, K.: Tolypomycin, a new antibiotic. V. In vitro and in vivo
antimicrobial activity. J. antibiotics 25, 16 (1972)
80) Cassani, G., Burgess, R.R., Goodman, H.M., Gold, L.: Inhibition of RNA polymerase by
streptolydigin. Nature, New Biol. 230, 197 (1971)
81) v.d. Hell, K., Krakow, J. S.: Inhibition of RNA polymerase by streptolydigin. Nature,
New Biol. 235, 82 (1972)
82) Reusser, F.: Titandamycin, inhibition of RNA polymerase. Infect. Immun. 2, 77, 82 (1970)
8a) Sokolova, E.V., Ovadis, M.I., Gorlenko, Zh. M., Khesin, R.B.: Localization of streptolydigin
resistant mutation in E. eoli chromosome. Biochim. Biophys. Res. Commun. 41,870 (1970)
84) Wu, G., Dawid, I.B.: Purification and properties of mitochondtial DNA-dependent RNA
polymerasr from ovaries of Xenopus laevis. Biochem. 11, 3589 (1972)
85) Follett, E.A.C., Pennington,T.H.: A direct effect of some rifamycin derivatives on the
morphology of mammalian mitochondzia. Exp. Cell. Res. 77, 47 (1973)
86) Meilhac, M., Typser, Z., Chambon, P.: Animal DNA-dependent RNA polymerases. 4. Studies
on inhibition by rffamycin derivatives. Europ. J. Biochem. 28, 291 (1972)
87) Busiello, E., di Girolamo, A., di Girolamo, M., Fischer-Fantuzzi, L., Vesco, C.: Multiple
effects of rffamycin derivatives on animal-cell metabolism of macromolecules. J. Europ.
Biochem. 35, 251 (1973)
88) Riva, S., Fietta, A. Silvestri, L.G.: Mechanism of action of a rifamycin dervative (AF/013)
which is active on the nucleic acid polymerases insensitive to rifampicin. Biochim. Biophys.
Acta 49, 1263 (1972)
89) Mcdoff, G., Kobayashi, G.S., Kwan, C.N., Schlessinger, D., Venkov, P.: Potentiation of
rifampicin and 5-Fluorocytosinc as antffungal ant~iotics by Amphotericin B. Proc.
Natl. Acad. Sci. 69, 196 (1972)
90) Kobayashi, G.S., Cheung, S.C., Schlessinger, D., Medoff, G.: Effect of tifamycin
derivatives, alone and in combination with amphotericin B, against Histoplasma
capsulation. Antllicrob. Ag. Chemother. 1974, 16
91) Battaner, E., Kumar, B.V.: Rffampin: inhibition of RNA synthesis after potentiation by
amphotericin B in Saccharomyces cerevisiae. Antimicrob. Ag. Chemother. 1974, 371
92) Begs, W.H., Sarosi, G.A., Andrews, F.A.: Synergistic action of amphotericin B and
rifampin an Candida albicans. Amer. Rev. Resp. Dis. 110, 671 (1974)
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strains with Amphotericin B plus rifampicin. Antimicrob. Ag. Chemothcr. 1974, 783
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Effect of Amphotericin B and rifampin against Coccidioides immitis in vitro and in vivo.
Antllicrob. Ag. Chemothcr. 1976, 406
9s) Medoff, G., Kwan, C.N., Schlessinger, D., Kobayashi, G.S.: Permeability control in animal
cells by polyenes: a poss~ility. Antimicrob. Ag. Chemother. 1973, 441
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multiple RNA polymerases. Proc. Natl. Acad. Sci. 69, 1702 (1972)
97) Wintersberger, E.: Isolation of a distinct rffampicin-resistant RNA polymerase from
mitochondria of yeast, neurospora and liver. Biochem. Biophys. Res. Comm. 48, 1287
(1972)
98) Venkov, P.V., Milchev, G.I., Hadjiolov, A.A.: Rffampin susceptibility of RNA synthesis
in a fragile Saccharomyces cerevisiae mutant. Antimicrob. Ag. Chemother. 1975, 627
48
99) Moreau, M.-C.: Inhibition of a vesicular stomatitis virus mutant by rifampin. J. Virol.
14, 517 (74)
100) Remillard, S., Rebhun, L.I.: Antimitotic activity of the potent tumor inhibitor Maytansine.
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101) Wolpert-Dufilippes, R.K., Adamson, R.H., Cysyk, R.L., Johns, D.G.: Initial studies on the
cytotoxic action of maytansine, a novel ansa macrolide. Biochem. Pharmacol. 24, 751
(1975)
1o2) Green, M., Gerard, G.F.: RNA-directed DNA polymerase properties and functions in
oncogenic RNA viruses and cells. In: Progress in nucleic acid res. and molec, biol. Vol.
14, p. 188. New York and London: Academic Press 1974
103) Wu, A.M., Gallo, R.C.: Reverse transcriptase. CRC Critical Rev. Biochem. 1975, 289
104) Gallo, R.C., Young, S.S., Ting, R.C.: RNA-dependent DNA polymerase of human acute
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polymerase activities of RNA tumor viruses. Nature New Biol. 229, I 11 (1971 )
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by a rifamycin derivative. J. Virol. 18, 445 (1976)
lO7) Pennington, T.H., Follett, E.A.C.: Inhibition of pox virus maturation by rifamycin
derivatives and related compounds. J. Virol. 7, 821 (1971)
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DNA-dependent RNA polymerase of vaccinia virus particles. J. Virol. 8, 133 (1971)
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vaccinia virus morphogenesis. J. Virol. 8, 225 (1971)
110) Becker, Y.: Antitrachoma activity of rffamycin B and 8-O-acetykifamycin S. Nature 231,
115 (1971)
Received October 4, 1976
49
Dr. Dieter Orth and Dr. Hans-Eckart
Radunz
E. Merck oHG, Pharma-Forschung, Abteilung Naturstoffe, 13-6100 Daxmstadt, Germany
Dedicated to Professor Theodor Wieland on the occasion o f his 65th birthday

(June 19 78)
Table of Contents
~
2.
Introduetion
Synthesis o f H e t e r o p r o s t a n o i d s
. . . . . . . . . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
52
55
55
56
56
59
63
2.1. 3-Thiaprostanoids . . . . . . . . . . . . . .
2.2. 3-Oxaprostanoids . . . . . . . . . . . . . .
2.4. 7 . O x a p r o s t a n o i d s . . . . . . . . . . . . . .
2.5. 8-Azaprostanoids . . . . . . . . . . . . . .
2.7. 9-Oxaprostanoids . . . . . . . . . . . . . .
2.8. 10-Oxaprostanoids
. . . . . . . . . . . . .
2.9. 11-Thiaprostanoids . . . . . . . . . . . . .
2.10. 11-Oxaprostanoids . . . . . . . . . . . . .
. . . . . . . . . . . . .
2 . 1 1 . 13.Azaprostanoids
2.12. 13-Thiaprostanoids . . . . . . . . . . . . .
2.13. 13-Aza-7-oxaprostanoids
. . . . . . . . . . .
2.14. 8,12-Diazaprostanoids . . . . . . . . . . . .
2.15.9,11-Dioxaprostanoids . . . . . . . . . . . .
2 . 1 6 . 9 , 1 1 - D i h e t e r o and 9 , 1 0 , 1 1 - T r i h e t e r o h o m o p r o s t a n o i d s
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. . .
. . .
. . .
. . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
. .
3.
Conclusions
89
4.
References
95
66
68
70
72
73
78
79
83
85
86
87
D. Orth and H.-E. Radunz

1. I n t r o d u c t i o n
Several reasons are responsible for the fact that prostaglandins - a group of natural
occurring substances - have been studied in biochemical, medical and chemical
experiments more intensively than any other group of substances in the last years 1-6).
There are few if any classes of compounds that have so many different actions
in cells, tissues and organs. Although they may have a wide range o f effects, they
nevertheless exhibit strict structure-activity specifity, thereby raising the question
how those relatively simple molecules can exert so many disparate effects.
The standard nomenclature for this family of readily released fatty acids from
tissues and organs and the synthetic prostaglandin.like compounds is derived from
the C-20 eyelopentane acid skeleton of the hypothetical prostanoic acid 1.
The numbering of the carbon atoms is, according to the nomenclature rules used
by the Chemical Abstracts 7' s), consistent with the numbering for fatty acids. The
compounds are typed depending upon the substitution pattern of the cyclopentane
ring and classified by the degree of unsaturation in the side chains; see Scheme 1.
E. J. Corey9) proposed the introduction of the term Prostanoids to designate
the family of natural prostaglandins and synthetic prostaglandin-like compounds.
Following this proposal the authors of this review want to designate the prostaglandinlike compounds which contain one or more heteroatoms in the prostanoic acid
skeleton at the place of one or more carbon atoms as Heteroprostanoicls. The
11
12
~
14
13
H
16
15
18
t7
20
19
Types (ring dependent)

OH
OH
Unsaturated classes (number of carbon-carbon double bonds)

O
OH
1
(mono)
Scheme 1. Classificationof prostanoids
52
OH
OH
2
(bis)
3
(tris)
Synthesesand Activityof Heteroprostanoids

following review is concerned with syntheses leading to these not naturally occurring
substances and comparison of their biological activities, as far as they have been
reported.
Before starting to present this fascinating chapter of chemistry, we have to
mention, however briefly, the biological potencies of the natural occurring
prostanoids so). The wide range of biological response they produce and the potency
of action they have was and is stir yet the main driving force behind the enormous
synthetic effort all over the world.
After their discovery and after yon Euler 11) had coined the term prostaglandins
which was in fact a misnomer, the prostanoids remained unnoticed for a long period
of time. The situation changed however completely, when S. Bergstr6m and his
co-workers 12) succeeded in isolating the active compounds and elucidating their
chemical structures. Before satisfactory studies of their pharmacologic effects
could be concluded, methods for the preparation of adequate quantities of relatively
pure prostanoids had to be evaluated.
Extraction and purification of prostanoids from animal tissues was insufficient
because the tissue levels are very low.
The development of bioeonversion processes from particular polyunsaturated
fatty acids ~3, ~4) yielded however sufficient amounts of various pro stanoids to
study their chemistry is) and biology and allowed preliminary clinical evaluation.
With the rapidly increasing knowledge of their pharmacologic actions the chances
of a practical medical use for induction of labor at term 16), for inducing therapeutic
abortion 17) for fertility control in man tS) and farm animals sg) for treatment of
thrombosis and for certain types of stomach ulcers 2~ rose,
The recent introduction of PGF 2 as Prostin Fza | is an example for the
clinical application of a natural occurring prostanoid.
Serious drawbacks of the natural occurring compounds however do not allow
their general use in therapy. The rapid metabolic deactivation 21 -2s) and the too
wide range of activity are the main problems which have to be overcome. The
development of other clinical applications awaits further progress in prostanoid
research and this meant a challenge to the synthetic chemists to develop chemical
total syntheses which could effectively compete with biosynthesis and which could
be easily modified in order to prepare analogs with improved selectivity and
stability to metabolic deactivation.
For planning and conducting these total syntheses the exact knowledge of the
chemical properties of the prostanoids is) which had been studied with biosynthetic
prepared prostanoids proved to be very helpful. Relatively soon it was discovered
how prostanoids can be metabolised and which parts of the molecules are the targets
of metabolic inactivation. Metabolism is mainly confined to the side chains.
~Oxidation of the acid side chain leads to the essentially inactive bis- and tetra-nor
acids and the oxidation of the 15-hydroxygroups results in the formation of inactive
15-ketones z6- 29).
The plans for effective synthesis therefore aimed at common intermediates
suitable for the synthesis of the natural prostanoids in high yield and for the
synthesis of desirable analogues with variations in the molecule around those areas
where metabolic variations occur.
53

Many groups of synthetic chemists have succeeded in the meantime in solving
the problem of total synthesis of the natural prostanoids by developing more or
less elegant routes 3~ 3Ob, 31 -33). The total synthesis developed by Corey 34)
however which has been modified in many ways 3s) is without doubt the most
successful and most commonly used synthetic method in the prostanoid field.
There is no other synthesis which allows both the preparation of all natural
prostanoids even in the optically active form 36' 37a, b) as well as the variation of
the structure of the prostanoids in a very wide range.
At the same time as the attempt to slowdown or prevent the fast metabolism
and the inactivation of prostanoids through chemical variations of the side chains,
was the hope also to find substances which act more specific and longer with lower
dosis. Many thousands of analogues have already been synthesized all over the
world by variation of Corey's method. The first successes to prevent the metabolic
oxydation of 15-OH as much as possible came with the introduction of the methyl
group in position C 1s 38) and/or C 16 39) and the partial substitution from the pentyl
chain (C17-C20) by aromatic ring systems4~ 41 ). For example, it was proved that
these compounds were no substrates for the 15-hydroxydehydrogenase (isolated
from human placenta) 42' 43). The/3-0xydation of the acid side chain can be
suppressed by substitution of the C3.methylengroup through an oxygen atom or
through alky1-44) or halogensubstituents4s) in the C3-position. In all of these
cases an extension of activity and a partial specifity of activity could be observed.
Parallel to these concepts with biochemical background other research groups
proceeded purely phenomenological. In this way the cyclopentane ring was substituted either by a cyclohexane ring 46' 47) or cyclobutane ring48) or by 5 or 6
ring aromates 49- 52). These analogues show no or only very minor biological
activity. Apparently by these analogues the special arrangement of the side chains
responsible for the activity is changed to much a degree that no or only insufficient
receptor bonding is possible.
N. H. Andersen eta/. s3- 5s) succeeded in their attempt to qualify the structureactivity relationship. Through their research results they proved the concept, that
the decisive prerequisite for the specific biological activity of each prostanoid is
to achieve easily a "hairpin" conformation in which both side chains are arranged
in a certain way to each other.
It can be seen from model observations that this hairpin conformation is only
then optimally attained, if both side chains are bonded to a cyclopentane, cyclopentene, cyclopentanone or cyclopentenone. Therefore the chances to obtain a
final molecule showing good biological activity seemed to be optimal, when the
prostanoid ring system is kept intact, while the ring substitution is varied.
This was experimented, for example, by introducing methyl groups into the
positions Ca s6) C9 57), Clo 58' 59), C1157) and C12 60-62). The Syntex research
group under Crabb6 tried to improve activity by shifting the 5.ringhydroxyl
group(s) 124) or by introducing an additional hydroxylgroup12s, 126). Finally concepts of synthesis were developed to vary the side chains.
Even though at the present time no conclusive judgement can be made about
the most favorable or acceptable changes with improvement of activity, nevertheless
the variation of the side chains seems to be the most promising.
54
Synthesesand Activity of Heteroprostanoids

In accordance with published and our own results the prostanoid analogues
should show biological activity, when the entire molecule conformation does not
differ greatly from that of natural prototype.
The substitution of one or more C-atoms of the prostanoids acid structure
by hetero atoms is one of the conceivable possibilities to fulfill this assumption.
In the following these ideas of synthesis will be presented and the attempt made
as much as possible, to find out by using published biological data, where the
introduction of hetero atoms caused qualitative and/or quantitative variations in
activity.
2. Synthesis o f H e t e r o p r o s t a n o i d s
2.1.3-Thioprostanoids
K. F. Bernady and co-workers 63) from the Lederle Laboratories have described a
useful procedure for the synthesis of dl-11-deoxy-3-thiaprostanoids based upon
the conjugate addition of E-l-alkenyl ligands from lithium E-l-alkenyltrialkylalanate reagent 3 to the sulfur containing cyclopentenone (2) 64).
It was found that the "ate".complex formed by treatment of alkenyl-lithium
with trialkylaluminum conjugatively transfers the alkenyl ligand to cyclopentenone
in relatively good yield. It is noteworthy that the total yield of the expected product
depends on the solvents used. The results - obtained in an analogous reaction indicate that the addition in hydrocarbons gives as by-products more 1,4-reduction
product than cyelopentenone polymer. On the other hand the cyclopentenone
derived polymer becomes significant in the more basic THF. The michael addition
3
0
/Nx.~..-~jSvCOaC2Hs
\ l
ether/hexane I :I
-40~
I) 80% HOAc,80~ 0,S hr
2) saponification
,~
Q
~"~-"~S~./~C02H
OH
O
-~/~S~CO2H
OH
55
obviously afforded only products in which the side chains are exclusively trans
orientated. The trans-configuration of the double bond in. the lithium alanate
reagent can be taken for granted. That means, that this process is accompanied by
retention of configuration. Protolytic work up of the reaction mixture, followed
by detritylationi dry column chromatography upon silica gel and saponification
gave in 27% yield the C ls-epimers, all-11-deoxy-3-thia-PGE 1, 4 and all-15-epi-11deoxy-3-thia-PGE 1, 5 in a ratio of approximately 45 : 55.
The biological activities of these new analogs are not reported until now.
The stereospecific synthesis of 3-oxa-prostanoids is described in the same publication
as for 3-thia-prostanoids by K. F. Bernady et aL 6a). The principle of this synthesis
is also analogous. In that case, the starting material is the corresponding 3-oxa-cyclopentenone, 6. Work up, followed by deprotection of the ether, chromatography
and saponification gave in 38% yield the free acids of dl-11-deoxy-3-oxa-PGE l 7
and its Cls-epimer 8, dl-15-epi-11-deoxy-3-oxa-PGE 1 , in a ratio of approximately
45: 55.
o
~'O~/'CO2C2Hs
severalsteps
41am.
"~/'~/O~CO2H + ~"~/~/~/O~CO2H
OH
OH
2.3. 7-Thiaprostanoids
J. Fried et aL 6s) reported a stereospecific synthesis of nat.-7.thia-PGFl,~ , 24,
ent-15-epi-7-thia-PGF t ~, 25, and racemic 7-thia-13-prostynoic acid 14. The
elaboration of the basic skeletal structure is exemplified by the synthesis of 14,
which is compatible with the additional functionality required for 24 and 25.
Reaction of cyclopentene oxide with mercaptohexanoate 9 in the presence
of sodium methoxide in methanol at room temperature produced the transhydroxyester 10. This ester was hydrolyzed to the acid 11, which was treated with
methanesulfonyl chloride in pyridine and afforded the trans-chloro acid 12 in 82%
yield. The fact, that no cis-chloro acid was obtained is an evidence for the formation
of a symmetrical episulfonium intermediate 13.
56
NaOCHa=
,,,
HS/~"~CO2CHa
98%
~ ..S~CO2CH3
~OH
10
11
13
98%
CHaSO2C1
. _ / "~.,q.
- - v.
A
, v .v C~TO 2 H
Li-C~C-C6Ht3
50%
12
...S.~~CO:H
~- ~C_~C_CoH~ 3
14
2H
15
The sodium salt of the chloro acid 12 was converted to racemic 7-thia-13prostynoic acid 14 by reaction with 5 equivalerits of 1-octynyllithium in DMF.
Catalytic reduction of 14 with excess palladium in ethyl acetate afforded the
cristalline 7-thia-prostanoic acid 15, m.p. 40-41 ~ This synthetic procedure was
successful also in the synthesis of 7-thia-PGFla, 25. Reaction of the protected
dihydroxyepoxide 16 with the anion of methyl-6-mercaptohexanoate 9, followed
by hydrolysis furnished the hydroxy acid 17. This acid was converted in two steps
into the bromo acid 18. The sodium salt of 18 was treated with (S).3.tert. butyloxy1-octynyllithium in DMF]hexane at room temperature to form, after chromatography, in 33% yield the mixture of diastereomeric acids. After conversion into
the methyl esters, debutylation and reduction with lithium alanate in boiling THF
the chromatographic separation gave the corresponding diastereomeric alcohols
20 and 21.
Debenzylation was achieved in 63% yield by converting the alcoholic groups
to their anions with sodium hydride in THF and followed by reduction with
lithium in ammonia-THF at -78 ~
A crucial step in this synthesis is the selective oxidation of the tetrols 22 and
23 but the application of a very interesting method, published by J. Fried and
J. C. Sih66) with platinium in aqueous acetone in the presence of sodium bicarbonate
gave 24 and 25, respectively, in 50% yield. The absolute configuration of the
products 24 and 25 were carefully determined by chemical and physical methods.
All of these three 7-thia-prostanoids show interesting biological activities.
7-Thia.13-prostynoie acid 14 is an inhibitor of the contraction of the gerbil colon,
and of the stimulation of adenylate cyclase in the mouse ovary caused by pros~
taglandin E t . This inhibition is as effective as for the 7-oxa-analog. Compound 14
57

OCH2Ph
QCH2Ph
..s 0
+ 9
"
75%
OCH~Ph
OCH2Ph
16
17
H
QCH2Ph
_~S~CO2 H
2 steps
l(S)
Li-C-C--C--CsHtt
~.t~ Bu
- Br
OCH2Ph
18
OCH2Ph
3 steps
=~Sc~CO2H
6CH2 Ph
OtmBu
19
OCH2Ph
QCH2Ph
OCH2Ph
/t
OH
OCH2Ph OH
20
OH
OH
21
OH
OH
22
HO
o.
OH
23
OH
24
m.p. 94-96~
[~]o + 5"50~ ( c 0,43; CH3OH )
OH
25
oel
[~x]D - 4,4 ~ ( c 0,27; CH3OH )
inhibits also the placental prostaglandin-dehydrogenase 5 - 1 0 times more than the

7-oxa-analog 67).
The 7-Thia-prostanoid with the natural configuration o f all chiral centers, 24,
stimulates the c-AMP synthesis in the mouse ovary, whereas 25, which possesses
58

unnatural configuration at four centers shows no remarkable activity in this model.
The heteroprostanoid 24 shows binding to a bovine corpus luteum receptor with
1/lOth of the affinity of PGFIa, while 25 exhibits 1/100th the binding of 24.
Both, 24 and 25 are inhibitors of the placental prostaglandin-I 5-dehydrogenase at
(/)so = 5,2/aM and (/)so = 8,8 t~M, respectively.
The structural similarity of PGF l a and the substance in which the 7-methylene
group has been replaced by ether oxygen, stimulated the research group of J. Fried
(University of Chicago) to look for a stereo-specific approach for the synthesis of
7-oxaprostanoids 6a). Fried was fascinated by the fact, that the stereochemistry
of the 7-oxaprostanoid and that of PGFt, ~ is fully analogous and that its geometry
differs only slightly because the C - O - C bond angle is somewhat larger (111,5 ~
than that described by the C - C - C bond (109,5 ~ The proximity of the 7-ether
group and the 9-hydroxylgroup enables the formation of a stable hydrogen bond
in the 7-oxaprostanoid which could be responsible for conformational changes.
The known differences in activity between natural prostanoids of the E and F
series which likewise differ in the same region of the molecule which was planned
to modify in Fried's work made it very interesting to study the biological properties
of these molecules.
The synthesis69) started with cis-cyclopentene-3,5-diol 7~ 26 which had been
obtained in a stereospecific reaction by addition of singlet oxygen to cyclopentadiene
and reduction of the intermediate cycloperoxide.
The oxydation of the cis-diol gave exclusively the all-cis-oxido-dio171) 27
which was converted to the cristalline dibenzylether 28. For the introduction of the
eight carbon chain with and without a 15-hydroxyfunction Fried and his co-workers
developed a very elegant aluminium-organic method. They succeeded in conversion
of the 3,5-dibenzyloxyepoxide by a very efficient alkynylation reaction into the
acetylenic alcohol 29. They could show that the opening of the epoxide ring had
occurred exclusively with the formation of the trans alcohol, as expected.
This intermediate was alkylated with tert.-butyl-~-iodohexanoate to the ester
30. Conversion to the acid 31 was achieved by cleavage of the tert.-butylester with
trifluoroacetic acid at low temperature. The triple bond was reduced to a trans.
double bond and simultanously the benzylether groups had been removed with
lithium in ethylamine, under formation of the desired 15-deoxy-7-oxaprostaglandin
F I ~ 32 in crystalline form. The cis-isomer was prepared by first reducing the triple
bond of compound 30 with palladium on barium sulfate to 33, removal of the
tert.-butylgroup with formic acid to 34 and debenzylation of the acid with lithium
in ethylamine to 35.
The fully saturated analogue was synthesized by catalytic reduction of 30 with
palladium on charcoal in ethyl acetate to 36 and hydrogenolysis of the benzylether
groups with palladium on charcoal in acetic acid to 37. The tert.-butylester function
was then cleaved with trifluoroacetic acid in hexane to 38.
59

PhCH:Q% ~,,OH
-----
?"r':::,o
---
H(~
26
",,,
PhCH20~
27
PhCH20r
OR
65% =
PhCH20'*a"
%
29
28
PhCn:Q~
29
"
-.~
Li/EtN
H2
30
R = terr.-butyl
31
R = H
32
Pd/BaSOa
PhCH20~
HO.
OR
PhCH20#
--
33
R = tert.-butyl
34
R=H
PhCH20~
30
Pd/C
~-
HOr
--
35
lOl
[-I"
HO~
~ "~O~O'~x'x
EtOA------~
HOAc
\~
2) CF3CO2H HO~
~.
PhCH20~
36
37
38
A A ~
. . .
i"
R = tert.-butyl
R = H
The introduction o f the 1S-hydroxygroup was possible by hydroxylation of

with SeO269) to yield the (+)-7-oxa-PGFl~ or its 15-epimer as a crystalline
substance 3 9 .
Although the 7-oxa-PGF 1~ obtained by the sequence mentioned above has the
absolute configuration of the natural prostanoids the substances synthesized were
racemic because the sequence of reactions was carried out on racemic material.
The synthesis o f ( + ) -7-oxa-PGF l and (+) -7-oxa-15-epi-PGF 1 and their
enantiomers was then started by Fried and his co-workers 72) by using the opening
of the meso-cyclopentene oxide 2 8 with a dialkylalkynylalane 4 5 which contained
the completely functionalized eight-carbon side chain in optically active form.
32
60
_ .o
o.
Lo"
OH
32
39
This led to the formation of two diastereomeric alcohols 47a and 47b which
were readily separated by chromatography.
This procedure required but one resolution of the acetylenic alcohol 40 which
then served to resolve the remaining chiral portion of the molecule. The resolution
of octyn-3-o140 therefore was the start of the synthesis of the optically active
7-oxaprostanoids. Reaction of the racemic octyn-3-ol 40 with phthalic anhydride
gave the phthalyl acid 41 which formed the crystalline salt 42 by reaction with
(-)-~-phenethylamine with the absolute configuration shown.
Optically active octyn-3-ol was obtained by first converting the salt 42 to the
ester acids 43 and then hydrolysis of the ester to compound 44. Optical purity and
assignment of the absolute configuration as S was established by methods known
from analogous compounds from the literature. Fried and his co-worker proved that
preparation of the tert.-butyl-ether derivatives and deprotection with trifluoroacetie
acid is possible without a trace of racemisation. This guarantees that the S (normal)
or the R (epi) 15-hydroxyprostanoids can be synthesized by using the resolved
tert.-butylethers in the alkylation reaction.
Dimethyl (S)-(.)-3.tert.-butyloxy.l-octynylalane 45 was therefore prepared
from (S)-(-)-3-hydroxy-l-ocyne44 with isobutylene in methylene chloride, followed
by lithiation and reaction with dimethylchloroalane. Condensation of all-cis-cyclopentane.3,5-dibenzylepoxide with this reagent in toluene formed the mixture of
diastereomeric butylethers 47a and 47b.
HO
(+_)-40
~ "~ o
~
~'OH
~ O
(
S"Phen~thylamine
(+)-42
(CH3hA1--~
RO
He
44
45 R = tert.-butyl
46 R= H
43
61

Debutylation gave the acetylenic alcohols 48a and 48b which could not be
separated by chromatography. The acetylenic alcohols could be prepared also
directly by reaction of the epoxide with the free alcohol 46 instead of the tert.butylether reagent 45.
Reduction with LiAIH4 in THF produced a mixture of olefins which was readily
separated by chromatography into the diastereomeric olef'mic alcohols 49 and 50.
The olefinic diols were alkylated with remarkable specificity for the ring alkylation
to 51 and 54 with tert.-butyl-co-iodohexanoate using dimsyl anion in DMSO.
Cleavage of the ester function to 52 and/or 55 and respectively debenzylation
furnished after column chromatography on silica gel crystalline (+)-7-oxa-PGF t
(mp. 65-67 ~ and 53 (+)-15-epi-7-oxa-PGF 1 56. Repeating the sequence of
PhCH20"
OH
PhCH20
.~
P h --C Ho~**,~
2
Hwr "//zOR
OH
PhCH20~ , , , , f
=
OH
49
2 8 + 45
28 + 46
tert.-butyl
47a
R =
48a
R = H
PhCH20~O~__
%
H
PhCH20''~
"
~ ~
Hd,,r, OR
PhCH20~O
~"~x'''-x'~
x%
.O
~' : H~v" x~" x'~
PhCH2 "~
OH
50
47b
R = tert.-butyl
48b
R = H
PhCH20~
IOI
HO~
50
_ _ .
PhCH20 -~
_~
OH
51
R = tert.-butyl
52
R=H
PhCH20,
Q
-----,- ~ . . ~ O . , . j . ~ . . ~ O
HO"
OH
53
~HO
49
PhCH20"
62
=
OH
54
R -- t e r t . - b u t y l
55
R = H
HO"
=
OH
56

reactions with (R)-(+)-3-tert-butyloxy-1-octynyldimethylalane instead of its antipode furnished (-)-7-oxa-PGFt a and (-)- 15-epi-7-oxa-PGFl 6.
On the way to 7-oxa-PGEl 12a) starting with the same all-cis-1,2-epoxycyclopentane-3,5-dio127 as used for the synthesis of 7-oxa-PGF-derivatives, Fried and
co-workers utilized some unusual selective reactivity of polyhydroxylated cyclopentanes.
The disilyl derivative of all-cis-l,2-epoxycyclopentane-3,5-dio157 was treated with
diethyloctynylalaneto afford after cleavage of the silylether function the trio158.
This triol was convered v/a the acetonide to the benzyl ether 59. Hydrolysis with
aqueous trifluoroacetic acid yielded the diol benzylether 60 which could be
prepared by an alternative route as well. This route proceeds v/a the monotrityl
epoxide 61 and benzylation to the trityl benzylether 62 and then reaction with
diethyl octynyl alane to the diolbenzylether 60 and the isomeric 1,3-diol.
Alkylation of the diol as mentioned above with tert-butyl-6o-iodo-hexanoate
gave the desired ether 63 and the isomeric ether, readily separated by chromatography.
The corresponding acid 64 was prepared with anhydrous trifluoroacetic acid.
This same acid was also obtained by selective debenzylation of the above mentioned
dibenzylether 30, which links this synthesis with that of 7-oxa-PGF1~. The keto
acid 65 was prepared with Jones reagent and the ketogroup then protected as the
ethylene ketat by converting the acid simultanously to the ester 66.
Alkaline hydrolysis led to the acid 67 which was debenzylated and reduced with
lithium in methylamine to the olefinic acid 68. Introduction of the 15-hydroxy
group 69 into the 7-oxaprostanoid was possible as in the PGF~ series with SeO2.
(+)-7-oxa-PGE1 70 and its 15-epimer were then obtained by removal of the ketal
group with trifluoroacetic acid and chromatography on silica gel columns.
The final product obtained ca. 10% of 7.oxa-PGA t the product of the ~-elimination
of the 11-hydroxy group, This ~-elimination took place also in neutral solvents
that means that this reaction is more easily happening than in the series of natural
E-prostanoids. Besides the above mentioned syntheses of 7-oxaprostanoids of the
E and F series Fried and its to.workers synthesized quite a lot of 7-oxaprostanoids
with different structures.
These syntheses will not be reported here and those who want to know more details
about the preparation of these derivatives can find these in the original papers
published by Fried and co-workers.
2.5.8-Azaprostanoids
Two very similar independent approaches to 1 l-Deoxy-8-azaprostanoids have
been published in 197573, 74)
Both synthetic routes started with methyl pyroglutamate 71 that means from a
starting material already containing the heterocyclopentanone system. Introduction
of the side chains was achieved in different ways. G. Bollinger and Joseph M.
Muchowski7a) prepared the half acid 73 by first N-alkylating the sodium salt of the
pyroglutamate with methyl-7-bromoheptanoate and then selective hydrolysis of
63
D. Orth and H.-E.Radunz
o~i(c..).
.o.
O'~I(cH3)3
HO"
o~"
27
"~"" ~ / ~ / ~ /
57
O-trityl
O-trityl
61
62
H%
x~'/'~OR
PhCH20''~
='~.~-~.~x
63
64
HO
~.~.~,,o...~
60
R = benzyl
PdlC=37
~
R = tert.-butyl
R=H
0
L~'Xo
O
xO
65
66
67
0
HO"~" "~
O
"]'OH~
69
64
_-
---~~
ff"x0
~
59
PhCH~O.,
7"I',,,,~o
.x~.Jx\\\
"
58
HO~--~to(O
30
PhCH20
R = (CH2)2-OH
R=H
H~
v
HO"
H
(3H
70
68
[""xn
O u ~O

0
_ _ _
0
71
7,
72
1~
o
1
o
OR -""'4" ~ , , , ~ O A c
75
76
74
a) R = H
b) R = OAc
the diester 72. Reduction of the acid 73 via the mixed anhydride led to the primary
alcohol 74 which was used for preparation of the enone 78 by Wittig-Horner reaction
similar to the way reported by Corey and others for introduction of the "lower"
side chain during the syntheses leading to prostanoids without hetero atoms.
The same intermediate 74 was synthesized by J. W. Bruin et aL 74) by an alternative
route. They reduced the ester function of the pyroglutamate with LiBH4 to the
primary alcohol 75a which was protected as the acetate 75b to prevent O-alkylation.
Reaction of the sodium salt of 75b formed with Nail in dimethylformamide, with
methyl-7.bromoheptanoate followed by methanolysis of the acetate function gave 74.
Oxydation of the alcohol 74 by Pfitzner-Moffat-oxydation or oxydation with
Collins reagent led to the unstable aldehyde 77.
Both teams reduced the enone to the mixture o f the C I s'epimeric alcohols
and separated the mixture by preparative TLC on silica gel or by column chromatography into a more polar 80 and a less polar 79 isomer with very similar spectral
data.
The relative configuration at Cls was tentatively assigned to be as in the natural
prostanoids to the more polar isomer by analogy with the chromatographic
behavior of similar derivatives of prostanoids reported in the literature.
In addition G. Bollinger and Joseph M. Muchowski took the chemical shift in the
laC NMR in which the a.isomer had resonance of carbon-13 at a lower field than
the 0-isomer as another strong argument for the correlation of the more polar
isomer to the a-series.
J. W. Bruin et ai. were able to prepare the 11-deoxy-8-aza-PGF2 , too by
alkylating 75b with methyl-bromo-5-heptynoate to 81, methanolysis of 81 and
partial catalytic hydrogenation of the triple bond to compound 82. The next
steps 83 -~ 84 and 85 were analogous to the procedure used for the synthesis of the
11-desoxy-PGEl -series.
The fact that the more polar ester or acid was more active in several biological
assays and that only the analogs 80 and 85 but not 79 or 84 had been shown to
65

0
74
--.. ~ - ~ ~ o ~
(CH3Oh,P_CH_C_CsHIIB ~
N A ~ / ~ O / "
0
77
78
Zn BH4
0
OH
OH
79
80
a) R = CH3
b) R = H
0
75b
~____/~
OAc
O'x1) methanolysis -~.~N.~X. ~ ~ O . . .

2) H2; Pd/BaSO~ ~ . ~ , . / O H
81
oxy tio
82
2) (CHaO)2P-CH-C-Cs HI~'
o.
"-~-="
OH
83
84
~ _~-=.~~OR
OH
85
a) R = CH3
b) R = H
be substrates for 15-hydroxy-prostaglandin dehydrogenase supports further that

the assignments mentioned above were correct.
J. Vlattas e t a t 7s) from the Ciba-Geigy-Corporation, USA, have considered the

replacement of the C 9.carbinol or C9-ketone functionalities by a sulfur atom in the
prostanoic skeleton.
66

The preparation of these compounds is an acceptance and extension of the
scheme previously used in the syntheses of natural according prostanoids by E. J.
Corey and co-workers76).
The michael addition of mercaptoacetaldehyde diethyl aeetal 8 7 and 9-cyanononenal
8 6 in the presence of triethylarnine gave the adduct 8 8 quantitatively. 1-Tributylphosphoranylidene,2-heptanone was reacted with the aldehyde and produced the
conjugated enone 8 9 , which ketalizated with four equivalents of ethylene glycol
and catalytic amounts ofp-tohenesulfonic acid in refluxing benzene to the bisdioxolane 90. Surprisingly, the isomeric bisdioxolane with the double bond at
positions Cl3 ' 14 was not detected.
A
.OC~H.
NC-(CH,),CH= CH-CHO+ HS'H~>(.OC;H;

86
NEt3/RT/15 hrs
quant.
87
:~.~CH2)6-CN
~ O),
, ~ ~CH
HsC 2
= X
88
X = 0
89
X =
CH-CO-CsHII
OC~Hs
L_/
90
x?
H ~#
91
X= OH; Y=OH
92
X= H; Y= OH
OH
93
94
~
OH
95
67

The intermediate 90 was treated with a small amount of PTS in acetone at room
temperature followed by chromatographic separation of the reaction mixture. The
enone 91 is the less polar epimer and its isolated yield, obtained by Vlattas et al.
is two times more than 92. The stereochemical assignments in 91 and 92 were
made by virtue of the difference between the resonances of their low field olefinic
protons. In the isomer with 11/t, 12~configuration, 92, the low field olefinic
proton appears 0,31 ppm downfield of the I 1 a, 12~-configurated isomer 91. The
upfield olefmic protons were in approximately the same position.
Reduction of 91, respectively 92, with zinc borohydride, separation of the C~sisomers, followed by hydrolysis gave (d/)-9-deoxy-9-thiaprostanoid 93 and (d/)11,15-epi-9-deoxy-9-thia-prostanoid 94. Oxidation of 93 with sodium periodate
produced a mixture of two epimeric sulfoxides 95, which were partially separated
by preparative thin layer chromatography. 7~
In another paper J. Vlattas and co-workers ) published an alternative synthetic
route leading to the preparation of racemic 93, as well as optically active forms
of 9,9.dioxy.9.thia-analog, 96.
The enantiomeric forms of 96 were obtained by resolution of the intermediate
enone 97. Esterification of this sulfone with R-(-)-~-methoxybenzeneacetyl chloride,
produced a mixture of diastereomers which were separated by preparative TLC.
Each diastereomer was converted to the enantiomeric alcohols by zinc borohydride
reduction, followed by chromatography of the Cls-epimeric mixtures. Saponification of the diolester gave the enantiomeric 9,9-dioxy-9-thia-prostanoids 96.
~'/~'/~/CO2CH3
OH
several stets
O
97
0 ~0
%I
1t
OH
96
2.7.9-Oxaprostanoids
The displacement of the C-9 by an oxygen atom has been planned by a research
group of Ciba-Geigy 78).
Their synthetic approach for the construction of the tetrahydrofuranone ring
started with 7-cyanoheptana179) 98 which was converted in the first step into
9-cyano-2-nonenoate 99 by reaction with sodium triethyi phosphonoacetate.
68

Addition of ethyl sodium glycolate to the/3-unsaturated ester 99 led to the formation
of the tetrahydrofuranone derivative 100. The same reaction was used for the
synthesis of 11-oxaprostanoids 8~ st). This intermediate was reduced to a mixture
of epimeric alcohols 101 which were protected as the tetrahydropyranylethers 102.
The ester group was then reduced to the primary alcohol function with LiAIH4,
the alcohol 103 oxydized with CrO 3 in pyridine to the aldehyde 104 and the
aldehyde condensed with 1-tributylphosphoranylidene-2-heptanone to the epimeric
prostanoid enones 105 and 107, which were separated by preparative TLC on silica
NaO~'ffO~-/
o
98
99
~N
1) NaBH4
"~"
~~/O~xXx~'~-~--~N
2iDHP/H~
~ ' x ~ O ~
RO O
100
LiAIH4
=-
101
R = H
102
R = THP
0
II
n-Buj)=CH-C-CslIIt
OH
~,~H
THPO
THPO
103
0
104
(O~x~X~-/~"
~ N + ( O ~ X x ~
105 R =THP
106 R = H
~N
107 R = THP
108 R = H
109
105
0
_
THPO
~H
110
HO"
OH
111
69

gel. The stereochemieal assignments in the formulas of 105 and 107 are based on
the data of the NMR spectra of the corresponding alcohols 106 and 108 which had
been obtained by hydrolysis of 105 and 107 respectively.
Compound 105 was used for the further synthesis and reduced to the mixture
of the C-15 carbinols 109 and 110 which were again separated by TLC chromatography. The polar isomer 110 was converted by hydrolysis of the cyanogroup to
the dl-9-desoxy-9-oxaprostaglandin El, 111, as a cristalline derivative.
Recently, an Indian work group reported another synthesis of 9-oxa-13,14dihydro-prostanoids127).
2.8. lO-Oxaprostanoids
The synthesis of 10-oxa-11-deoxyprostanoids that is of prostanoids with a 7-1actone
structure has been reported in the patent literature 82) and from a research group
from the Research Triangle Institute 83). The latter synthesis started with diethyl2-(3-cyclooctenyl)-malonate 112 which can be prepared according to the literature
84, 8s) by a treating the sodium salt of diethylmalonate with 3-bromocyclooctene
or 1,2-dibromocyclooctane.
Reduction of the compound 112 with LiAIH4 afforded 2-(3-cyclooctenyl)-l,3propanediol 113 which was oxydized by reaction with ozone to the acid 114.
This acid is a mixture of cis- and trans-isomers. By esterifying the acid to the
methyl ester with diazomethane a two compound mixture is formed, whereas ester
formation under acidic conditions (MeOH/HCI) gives a single compound. This
single compound 115 is thought to be the trans.isomer, because isomerization of
the mixture of esters obtained by reaction with diazomethane to a single compound
is apparently possible by an esterification relactonization mechanism by treating
the mixture with acid in methanol. These results are consistent with the formulation
of the ester 115 as the trans-isomer.
The intermediate carboxylic acid side chain in compound 114 is one methylene
unit shorter than that of the natural prostanoids, therefore chain elongation
OH
o~/
~o
112
EHo
Ho H
o j
113
114
70

starting with the carboxyl function had been necessary. As a first step on this way
the primary alcohol function was protected by reaction with Ac20 in pyridine.
The resulting acetate 116 is formulated as the trans.isomer, too. The carboxylic
group was then converted by selective reduction with diborane to the alcohol 117.
The alcohol function was converted to the nitrfle 119 via the tosylate 118 and displacement of the tosylate group with sodium cyanide. Methanolysis led to the
methylester 120 because the acetate moiety was not cleaved under these conditions
(MeOH/HC1). Hydrolysis with base yielded the deprotected lactone acid alcohol
121, which was purified by converting it into the methylester 122.
The primary alcohol function was then used for the introduction of the second side
chain via the aldehyde 123, the enone 124 and after reduction of the enone the
mixture of the epimeric alcohols 125 and 126, which could be separated by pressure
column chromatography. Structural assignment is not given.
Reaction of the enone with methyl magnesium chloride gave rise to the tertiary
alcohol 127 (which is a double racemate).
OH HCI/MeOH o O ~ \ ' ~ ~
-~OH
114
O'"
115
0~-~,,"~~'~0
k...A,,,,~OAc
___O ~ O A
117
118
119
120
116
~..f~/~R
___,..ok...j,~/)
HO~\\x\x
~'~"'~O
R = OH
121 R = H
122 R-- CH3
R = OTos
R = CN
R = CO2CH3
O
0
O, 9
0
125
O
I
O,
u,
H
126
*k
I
HO CH3
127
71

2.9.11-Thiaprostanoids
The synthesis of some 11-thiaprostanoid analogs is described by a Syntex research
group 86).
Tetrahydrothiophenone 1 3 0 was prepared in 52% yield by addition of sodium salt
of methyl thioglycolate 1 2 9 and m e t h y l - 4 - t e r t . - b u t o x y b u t - 2 . e n o a t e 128.
O2CH3
+ NaS'~'~CO2CHa
H2-O-~Bu
128
0~'~~CO2CH3
\S.-~O-~Bu
129
130
i) 1-(CH2)6CO2CHs/NaH._ 0~"~(CH2)6COOCH~
2) Lil
\S.---L..IO-t~Bu
1) reduction
2) Ac20/Py
im
3) HF/H20
131
OAc
CH2)6COOCH3 1) oxidation
OH
OAc
~x'~\ \~(CH2)6COOCHa
2) Wittig-react. S ~ s H t ,
I
132
3 steps
CH2)6COOCH3
'S.~~.CsH,,
O-CH-CH3
1) oxid
OC2Hs
134
135
""
136
0(:~" ' ' ~ C O ~ H

135
137
OH
131
several steps
~C02H
-~
OH
138
72
<CH2)6COOCHa
'S~.~/~CsHI,
OH
2) ~
soponific.
O
133
'

Alkylation of 130 with methyl-7-iodoheptanoate and subsequently decarboxylation
with LiI in DMF under reflux gave the ketoester 131 in 24% yield. Reduction with
sodium borohydride, acetylation, and cleavage of the tert.-butyl protecting group
with 48% aqueous HF and THF afforded the alcohol 132. The remaining steps in
the synthesis were completed by well known methods: oxidation, followed by
Wittig-Horner-reaction gave the enone 133 in which is assumed, that side chains at
position 8 and 12 are orientated in the more stable trans-position. Zinc borohydride reduction, protection of the allylic alcohol, saponification of the acetate
group, renewed oxidation and cleavage furnished v/a 134 the methyl ester of
15-hydroxy-9-oxo-11-thia-prost-13-enonic acid 135. Hydrolysis of the ester group
gave 15-hydroxy-9-oxo-11-thia-prost-13-enoic acid 136. The oxidation of 135
with one equivalent of m-chloroperbenzoic acid in methylene chloride produced
the mixture of isomeric sulfoxides 13Z
For the synthesis of the I l.thia-prostanoid analog of the F-series 138, the ketone
131 was reduced with potassium tri-sec.-butylborohydride (K-Selectride),
followed by using the methods described above.
Both prostanoids 136 and 138 show a small activity (ca. 0,005 times the
activity of PGE2) in the gerbil colon assay. The activities of the corresponding
sulfoxides or sulfones are not reported.
2.10. 1 l-Oxaprostanoids
The synthesis of 11-oxaprostanoids, that is the displacement of the carbonatom
11, which bears the 11-hydroxygroup of the E and F prostanoids by an oxygen
has been studied in several research laboratories all over the world. At least four
groups have published their results in 1974 and 1975.
The first synthesis reported was that of a Syntex-group s~ They used the well
known addition of the anion of methyl glycolate to or, &unsaturated esters 87- 90)
for the construction of the oxacyclopentane-system.
The synthon they used for the reaction with the anion of methyl glycolate
to the tetrahydrofuranone derivative 141 was methyl-4-tert.-butoxybut-2-enoate
140. This compound had been prepared out of the corresponding acetylenic ester 91 )
139 by partial hydrogenation. The/3-ketoester 141 was then C-alkylated by treatment
of its potassium salt with methyl-7.iodoheptanoate in DMSO and decarboxylated
with lithium iodide in DMF to the ketoester 142 which was claimed to have the more
stable conformation with trans side chains.
The intermediate primary alcohol 143 was obtained in three steps out of 142. The
further synthesis went v/a the aldehyde 144 (obtained as the hydrate), the enone
145 prepared by previously applied methods, to the mixture of the alcohols 146.
The corresponding 9-ketoprostanoid 148 was obtained in three additional steps
v/a the protected intermediate 147 and in another two steps, as a mixture of the
15-epimeric alcohols 148. Separation of the epimeric alcohols was possible by
column chromatography on silica gel.
Another similar route using the same principle of addition reaction was studied
by two independent groups al, 92).
73
I ) KOH
2) I ~ O
3) Lil
139
140
141
9O.,...x...,~..T~
AcO
3) CF3CO2ff
O
OH
142
AcO
143
O
0
AcO
(CHaO)2-P-CH-C-CsHIz
O
o/Zn(BH~2
"o-~OH
OH
0
144
AcO
145
~ \ \ \ \ V ~ . ~
OH
~ ~/.~\\~.~,.+~.~..,+.u~t-)"
IOro....
1) "~'O/~/H ~
2) CrO3/Pyridin-
1) H(~
2) OH~
O...~
146
O~
0
147
OH
148
Both used methyl-4,4-diethoxycrotonate 149 as reagent for the synthesis of the

tetrahydrofuranone derivative 150. The carboxylic side chain however was introduced by different ways.
One group 92) chose the way analogous to the afore mentioned synthesis i. e.
C-alkylation of the ~ketoester 150, hydrolysis and decarboxylation to 151.
Interestingly enough the acetal function was not cleaved under these strongly
acidic conditions.
As mentioned in the other publication al), too, the cleavage of the acetal function
is however readily achieved by reaction with mild acid yielding the desired aldehyde
when the keto-function of the ring is reduced before.
Using the unprotected aldehyde function as mentioned above for construction of
the remaining side chain, this approach leads in analogous manner as from 144
to 1 l-oxa-11-deoxyPGE 1. The other group al) used the ester function in compound
150 for the construction of the carboxylic side chain by a novel method. This was
74
done v~ the compound 153 by reduction of the ester to the aldehyde 154 and
conversion of 154 to the mixture of the cis and trans vinylthioethers 155. Three
additional steps were necessary to yield the elongated aldehyde 156, which was
converted to the intermediate 157 as usual.
The construction of the remaining side chain was possible in four steps v/a
158 to the mixture of the 15-epimeric alcohols 159 which were separated by
preparative TLC on silica gel. The isomers were separately reacted with dihydropyrane, the acetoxygroups removed by methanolysis to 160 and the end products
161 and 162 prepared by two or three additional steps.
o/oO
.i 1) KOH
2) 1 ~
3) H e
----
150
149
tl
"
O,
~'-l*~'~/~O
~1
151
1) NaBH4
2) DHP/H ~
~ O
152
153
~ Diba___~h--"\O~o~. ~ PhaP=CHSPh___
R =H
154
R = THP
THPO~
2) AI/Hg
3) K:CO3
155
1) He
156
H
157
0
O~,,," ~'~'~'~
v
O
O.~
" 1) Ac20
AcO
\~
~,,'~ " ~ - ~ . ' ~
o.
158
159
.oxydation__
2) H+
\
1)
OTHP
160
OH
3) O H -
~
O
O
~
OH
161 R = H
162 R = CH3
75

Structural similarity with furanose sugars was the common idea for two independent groups which published the synthesis of optically active 11-oxaprostanoids93- 9s).
The first group 93) chose an approach by which the introduction of the "upper"
side chain was achieved by reaction of the optically active epoxide 165 with the
sodium derivative of diethylmalonate to the mixture of the isomers 166 and 167.
The desired isomer 166 was isolated in 20% yield by chromatography on silica gel.
The stereocontrolled opening of the epoxide which had been prepared out of
163 via the mesylate 164 was the prerequisite for the correct configuration of the
prostanoid side chains in compound 171.
Compound 166 was converted to the diol 168 which then was oxydised with
periodate to the aldehyde 169.
The aldehyde function of 169 was used as usual for the construction of the "lower"
side chain to compound 1 70 which seems to be a suitable intermediate for the
synthesis of 1 l-oxaprostanoids.
The conversion of the intermediate 170 to optically active endproducts as e. g.
171 has not been published yet.
The other route 94) to 11-oxaprostanoids from branched ribofuranose sugars
started with D-xylose which was converted by a method published by W. Sowa 96)
to 1,2-isopropylidene-5-0-trityl-~-D-erythropentofuranos-3-ulose 172.
Condensation of 1 72 with the potassium salt of trimethylphosphonoacetate
followed by hydrogenation gave compound 173, which had trans configurated side
CH3
_
NaOMe
o-/- W
O~.
~/_.~ ~
OR
EtO
O*2-'-OEt
165
163 R = H
164 R = mesyl
HO
166
//--OEt
O
oH/
168
169
3 steps
HO' k .,,,
</~w "'CO2Et
76
O/,]._.OEt
HO~...l,~xxi . ~ O Et
,0-%_o
O
.___,,_
8H
170
1 71
O~OE t
167
Trit y1-OCH2-..~O'N._O
~'
oH,
"O'\CH3
steps
0 Trityl
CI-...,/0~__ 0
Hz ~
~::CHs
173 R = CO2CH3
174 R -- (?Ha-OH
1 75 R = CHO
1 72
TrityI- OCH~.x/O',,r_._O
3 steps
2 steps
CHs
II
O
v CH3
176
O
177
CH~ 0
II
CH
. ~ ".r__~,,"~./'~-~'~/-~OCH Zn(BH4)z
~ ( A S v / / ~ O C H
O
1 78
'
OH
1 79
chains as proven by its NMR spectrum. The carboxylic side chain was constructed
in several steps by the sequence 1 7 3 - 1 7 6 .
The deprotected compound 177 was then used for the introduction of the remaining side chain 178. Compound 179 was obtained as a mixture of epimeric
alcohols and can be regarded as an 11-oxaprostanoid derivative in optically
active form.
Another synthetic route published by the same authors 9s) started from D-glucose
which served for the preparation of the known 1,2,5,5-di-O-isopropylidene-D-ribohexofuranos-3-ulose 180. For compound 181, which had been synthesized by
condensation o f 180 with triethylphosphonoacetate and hydrogenation, the alloconfiguration was proven by NMR studies. Selective hydrolysis and acetylation
of the resulting diol gave the diacetate 182 which was converted under acidic conditions to the lactone 183. The free additional hydroxy group was then eliminated
by acetylation with p-nitrobenzoic acid chloride conversion via the furanosyl
bromide (HBr in CH2C12) to the phenylthiofuranoside (C6H s SK in EtOH), and
desulfurisation to the diacetate 184. Deacetylation and oxydation (NaJO4) led
to the aldehyde 185. The "lower" side chain was constructed as usual resulting
in a mixture of epimerie alcohols which were converted to their tetrahydropyranylethers 186. The remaining side chain is built up in two steps as in the case of the
natural prostanoids 187. Preparative layer chromatography on silica gel resulted
i n isolation of the more polar compound in pure form.
This isomer has been tentatively assigned the 15-S-configuration in analogy to
the TLC-behaviour of the natural prostanoids.
77
CH3
O~
2 steps CH3~"q~,.e/Ox,r....O
OO ~ ~ O ~ c
o H
CH
i 3
3
C2~sO ~ _ . ~ C H 3
---o~/__/
u CH3
O
180
Ao
2 steps
-- CzHsO
AcO
0~r__/
181
H 'ste
q,~O
O
183
O
H3
v CH3
182
oA,
oA.
OAc
OAc
184
185
~L ,
3 step,~._~.
2 steps ~~- C
OH
186
187
2.11.13-Azaprostanoids 97)
The synthesis of these heteroprostanoids in which the 13,14-double bond which
is both relevant for activity and metabolic degradation of the natural prostanoids
isaltered by introduction of a tertiary nitrogen at position 13 started from the
well known epoxide 18898).
Reaction of this epoxide with methylamine yielded the mixture of position
isomers189 and 190. The isomers were separated by column chromatography on
silica gel, and the structures correlated by studying their NMR spectra.
N-alkylation of 189 with either 1-bromo-2-tetrahydropyranyloxyheptane or
1-bromo-2-heptanone was not suited for the further synthesis.
Alkylation with 1-bromo-2-tetrahydropyranyloxyheptane gave only low
yields of 197. The alkylation with 1-bromo-2-heptanone resulted in formation of
the tricyclic compounds 196 instead of the desired compound 195.
An alternative route, opening of the epoxide with N-methyl-2-hydroxyheptylamine99)
to the mixture of the position isomers 191 and 192 was developed further, because
the separation and structural correlation with NMR was possible also in this case.
Cleavage of the acetal function to the hemiacetal 193 was achieved by treatment of
191 with perchioric acid. The last step of the synthesis was the introduction of the
remaining side chain in analogy to the method developed by E. J. Coreyl0 ~ in the
case of the natural prostanoids.
The target molecule 194 was obtained as a mixture of the 15-epimeric alcohols
which could not be separated further.
78

01
o\
01
0f
__..
~////OH
HN~.cH ~
190
O~
189
195
7 \
196
OR2
0
C02H
H
CH3 OH
OH CH3 OR1
188
194
191
193
197
Rt = H;R2 =CH3
Rt = H; Ra = H
Rt = THP; R2 = CH3
Oj
~'%'OH
C H 3 / N ~
192
We have investigated in our laboratories I 0s) the total synthesis of heteroprostanoids
which contain a sulfur atom in the lower side chain. We assume, that a small
enlargement of the distance between the hydroxy groups at positions C 11 and Cls
should change the biological activities of these new compounds. It is noteworthy in
this connection that from the displacement of the C 1s-hydroxy group to position
Ct6 in the PGF2-series, R. Pappo et as lOl) obtained compounds which are extremely
potent gastric antisecretory agents 1~ We have investigated an efficient synthesis of
13-thia-prostanoids, based on the michael addition of corresponding substituted
mercaptans to the well known 7-(3-hydroxy-5-oxo-cyclopentenyl)-heptanoic acid
201 in the presence of a basic catalyst. The preparation of the cyclopentenone
building block is frequently described in the recent literature 1~ 1o4)
79

Most syntheses start from 7-(5-oxocyclopentenyl)-heptanoic acid 198 followed
by introduction of the hydroxy group at position C a in two steps. We have repeated
this synthesis and found, that the change of the very sensitive allylic bromide 199
into 201 is possible on different ways. The SN2.reaction succeeds with silver acetate
(way A), as well as with calcium hydroxide (way B) or in a phase transfer reaction
with BuaN+Br- (way C) (Scheme 2).
Treatment of 200, obtained by way A, with lithium hydroxide in methanol, containing a small amount of water, at room temperature 1~ gave the expected synthon
201 in pure and excellent yield [m.p. 55-58 ~ (ether)].
The lower mercapto side chains were mainly prepared by formation of epoxides
starting from the corresponding ketones or aldehydes, respectively1~ Treatment
CO~H NBS/CCI4_ ~
198
O2H
199
CO2H LiOH
OAc
C02H
HO
200
Rl
2~O
R
201
Scheme2.
Rl~/Ok
R t. OH
------.-~ /---- -----~ 2 ~ x
R2
R
SH
+
HO
HO
CH3
bPTo=112-114~
201
202
O//C02H
Oe/~.-'~/~../~/CO2H
HO'
203
8O
12
15
HO~"CHa
204
Scheme3.

of the terminal oxiranes with H 2 S in presence of amines l~ give the desirable
&hydroxymercaptans in near quantitative yields (see Scheme 3).
The anions of these mercaptans, successfully generated with steric hindered amines
underwent smooth addition to the tx,/3-unsaturated five membered ring ketones
201. Under defined reaction conditions we obtained 203 as a major product when
202 was added to 201. Column chromatography on silica gel of the crude product
gave besides 203 a small amount of the isomeric compound 204.
The stereochemical structures assigned to the obtained 11-thiaprostanoids
203 and 204 has been supported by the results of IR and MS-spectra, and especially
by NMR double resonance technique. The most notable difference in the l H-spectra
of both epimers is the differentiated fine structure of the signals for the protons at
positions C 11. In isomer 203 this proton appears as quartett, centred at 4,26 ppm
(Irradiation experiments show the following coupling constants: J1 n, t 2 = 9,5 Hz,
Ja, 12 = I 1,0 Hz). In contrast, compound 204 shows for the same proton a triplett,
centred at 4,36 ppm (Irradiation experiments show as coupling constants: Jt 1, 12=
= 3,5 Hz, Ja, 12 = 11,0 Hz). These findings are in good agreement with those
presented in the NMR spectra of prostanoids E l and 11-epi-E 1 by M. Miyano et at
109-111). In addition, a convenient preparation of 13-thiaprostanoids of the
F-series has been generated from the 13-thia-E-prostanoids. 203 was converted into
the isomers 205 and 206 by reduction with sodium borohydride or other complexed
hydrides.
OH
OH
~-.T-'~CO2H
~--.-[-'"~I/~/CO2
203
~
205
HO...-'X""'~S~
206
The assignment of the stereochemistry of both compounds based on NMR

spectral data and their different behaviour on thin layer plates (silica gel), impregnated with boric acid 112). Only isomer 205 is able to form a cyclic boric ester,
therefore 205 runs faster on impregnated plates than 206.
An unresolved problem is the differentiation of the Cls-isomers. It is impossible to
differentiate these epimers whether by NMR-technique nor by thin layer chromatography.
In order to synthesize the enantiomeric forms of the methyl ester of 203 we
started from the described methyl ester of 7-(3(R)-hydroxy-5-oxo-cyclopentenyl)heptanoic acid I 13) and the optical active lower side chains.
Scheme 4 presents the route of total synthesis of optical active mercaptans 210
and 211.
The racemic aminoalcohol 20 7 has been resolved into both enantiomeric forms with
(D)-(-)-mandelic acid. Liberation of the amines, following by quaternization with
methyl iodide, treatment with silver oxide, and heating in water gave the optically
active oxiranes 208 and 209.
81
D. Orth
a n d H.-E. R a d u n z
)<..CH3
n-CsH1,
I) NaHSO3
2) KCN
HO CN
><.CH3
n-CsH,1
b'P'2$mm= 87-90~
HO
/ ~ ' ~ _ _ NH 2
n-CsHxt
CH3
207
CH3
R~Ha
s:
n-CsHl,'~
20
and CsHn / ~3,,,,~
--
[c~] 366- + 14,5
20
[~1366- 12,6 ~
(c = 5,0; CH~OH)
- -
(c = 5,5; CH3OH)
208
209
1H2S
IH2s
CH3 .~ .
HS"" / ' ~ ="~S

I 2n
":s n'~
OH
- -
210
Scheme
CH3
~..
-= 5 ~ n - " s n ~ l
"~
OH
[~ls~6- " 3 , 8 ~
(c = 7,8; CH3OH)
20
~
I-IS"
~ o _ +2,8 ~
[o~]3~-
(c = 5,0: CH3OH)
211
4.
These both key intermediates were opened with H 2 S in the presence of diisopro.
pylamine. This reaction is known to proceed with full retention of configuration.
Therefore we assume, that the obtained thiols 210 and 211 are of the assigned
absolute stereochemistry. The optical purity of each enantiomer was directly
determined from the relative peak areas and senses o f nonequivalence of the
resonances of enantiotopic nuclei in chiral solvent, e. g. Eu(TBC)3. We observe
optical purities for 2 1 0 p = 85% and for 211 p = 75%. The addition of 210 to the
optically active 212 gave after column chromatography the desired 8 R, 11 R, 12 R,
15 S- 13-thiaprostanoid E 213.
H__~
~ C O 2 C H s
H6
OO2CH3
no
[a]~~ = +16,8~
212
82
+ 210
[al~~ = -64~
213

Some of these new heteroprostanoids indicate very interesting biological
activities* 14). The E-type analog 203 is a potent vasodilator, its effect in blood pressure (cats i.v.; dogs i.v. or orally) was greater than that of PGE 1 . The unnatural
configurated analog 204 showed only a small activity on blood pressure in cats
and was inactive in dogs. Among the prostanoids studied, 205 as representative
compound of the F-series showed remarkable activity in the antifertility-test
in intact hamsters.
2.3. 13-Aza-7-oxaprostanoidsl 1s)

These prostanoids were synthesized starting with the well known dibenzyloxyepoxide 68) 214. The preparation of 13-aza- 13-N-methyl-7-oxa. 15-desoxy PGF 1~
from this starting material was possible by two different routes.
The first route was rather straight forward introducing first the nitrogen containing
side chain by reacting the epoxide with methylamine in methanol at 120 ~ under
pressure to the amino-alcohol 215 and N-alkylation of this intermediate with
1.bromoheptane in ethanol to the tertiary amine 216. This same product was
prepared directly by reaction of the epoxide with N-methylheptylamine in ethanol
with a catalytic amount of hydrochloric acid at 120 ~ under pressure. O-alkylation
was performed according to the method used by J. Fried (~9) that is reaction of 216
with tert..butyl.o>iodohexanoate (Nail in DMSO). Compound 217 was charac-
PhCH20~f..~\,xO
H'~k''x'N~
PhrH2O-.~
-------*-PhCH20(.
~
NO~''~/~'/OR
,
PhCHaOv
I
CH3
221 Rt = C~Hs
)I I
CH30
220
PhCH20~,.~,,uO
.__.~x,x..~k~. PhCH20~
(~OH
PhCH20"~'"
214
PhCHlO ~
PhCHzO~..],\\,OH
NH
l
CH3
<-~%~
PhCH20. x,,OH
r~,,~'~ "I ~ ~
CH30THP
PhCH~-
215
219
PhCH20~<
~-]'~
PhCH20#~X'1~/~~
CHa
216
222 Ri = H
l/"
HO~
__~
PhCH,O~X~"~'~~
CHa
217
He~
0
~
CH 3
218
83

terized as the hydroiodide (m.p. 7 8 - 8 0 ~
The removal of the benzyloxygroups
was carried out with the free base by reaction with BF3-etherate in benzene. This
reaction sequence led to the PGFla.derivative 218 as the free acid, because the
tert..butylester grouP was cleaved under the reaction conditions simultaneously.
This reaction sequence worked quite well for the synthesis of 15-desoxy-13-aza7-oxaprostanoids. The synthesis of 15-hydroxderivatives however was not possible
by this route. Reaction of 215 with 1-bromo-2-tetrahydropyranyloxyheptan gave
the N-alkylated product 219 only in very low yield. N-alkylation of 215 with 1bromoheptanon-2 resulted in formation of bicyclic compounds 221 when the
reaction was carried out in ethanol or 222 in tetrahydrofurane (instead of 220).
Therefore a second route using the N-protected intermediate 223 which was
prepared by reaction of 215 with tert.-butyloxy-carbonylazide was started. The
O-alkylation of this compound was easily possible by reacting it with ethyl-~obromohexanoate and silver oxide in DMF to 224. The t-BOC protecting group
in 224 was then removed smoothly with trifluoroacetic acid to the unprotected
amine 225.
The amine 225 was the common intermediate for the synthesis of both 15-deoxyand 15-hydroxy derivatives.
PhCH2O,
215
PhCH20,
~
~ ~H 3 ~
"M.,,A,,,N,A--
~
" x ~~x , / CH3
O ~ "0
PhCH20~
~
O~O
223
<
~N/H
3
224
225
The former prostanoids were synthesized by reaction of 225 with 1-bromoheptane to 226 and cleavage of the benzylether protecting groups to 22 7 which
was converted to the free acid 228 by alkaline hydrolysis.
The way to the 15-hydroxyderivatives was opened by N-alkylation of 225 with
1-bromo-2-heptanone in ethanol to 229a in good yield. The reaction product 229a
(free base m.p. 131 ~
m.p. 77 ~ was then used for the preparation
of the 15-methyl- 15-hydroxy and the 15-hydroxyderivatives.
The latter compounds were obtained as a mixture of the 15-epimers 230 by re-
PhCH20~..
HO~
225
PhCH20"~
~
RO"
[
CH3
226
84
N~ ~ ~ /
I
CH3
227 R -- C2Hs
228 R = H
Syntheses an d Activity of Heteroprostanoids

duction of the 15.keto-group with NaBH4 in methanol and then cleavage of the
benzylethergroups as usual (BFa-etherate in benzene). Separation of the epimers
232 was possible by chromatography on prepacked columns with silica gela).
Designation of the 15-hydroxy compounds to either the natural or epi series was
not possible.
Chromatographic separation of the 15-epimers was also possible when the free
acid derivatives 231 bearing the dibenyloxy protecting groups were used. Separate
treatment of the derivatives 231 with BF 3.etherate in benzene gave the two racemic
15-epimers. 233
Reaction of the 15-ketoderivative 229b as.the free acid with an excess methylmagnesiumbromide in ether gave rise to the mixture of the 15-methyl-I 5-hydroxy
compounds 234.
Chromatographic separation was obtained also in this series by using prepacked silica
gel columns. Removal of the protecting groups yielded the two racemic endproducts
235. As in the case of the secondary alcohol derivatives correlation of the separated
epimers was not possible.
PhCH~Q
225
"
tOi
~
PhCH20"~
229a
"
"
"
[ [[
CH30
"
x
~
PhCH2(~"
R = C2Hs ~
229b R = H
PhCH2Q~
"N
'd
iOi
N
I
CHa OH
CHa OH
230
R = C2Hs
232
R = CaHs
231
R = H
233
R = H
PhCH~q
O~OH
0
HQ~.
.
PhCH,0:
~N
~
234
HO~~
cNH3
[ OH
~
235
2.14. 8,12-Diazaprostanoids
These compounds have been published in the patent literature 116).
One of the syntheses mentioned there started with 3-pyrazolidinone 236,
which was protected as the benzyloxycarbonyl derivative 237 in order to obtain
selective N-alkylation at position 8 yielding compound 238 after removal of the
protecting group. Addition of the resulting amine 238 to l-octyne-3-one 239
formed the enone 240 with the complete diazaprostanoid skeleton. Katalytic
a) Fertigs~ule Kieselgel 60, GrOt~e A, E. Merck, Darmstadt.

85
D.Orth and H.-E. Radunz
0
~I~H - -
0
~/~IH
2 36
0
I~
~
~NH
237
238
.~ ~ ' ~ N ~ O R
2steps_=_ ~ " N ~ O R
2 steps__
~ N ~ O H
OH
240
241
reduction of the enone and reduction of the ketogroup to the mixture of alcohols
241, were the last steps on the way to 8,12-diaza- 11-deoxy-PGEo and its 15-epimer.
2.15.9,1 l-Dioxaprostanoids
These compounds with the structure of cyclic acetals or cyclic carbonates have
been synthesized by I. T. Harrison and V. R. Fletcher 117). The compounds represent prostanoids in which oxygen heteroatoms replace hydroxymethine or keto
groups in the eyclopentane ring.
The key intermediate for the construction of the dioxacyclopentane moiety was
the dio1247 which has been synthesized from the trans-olefinic ester 242. This
olefinic ester had been used by the same research group for synthesis of other prostanoids published 19721 l a). The preparation of 246 had been performed by reaction
0~ ~
~~0
HO~VVV~
0~"
0
242
243
0/
"
245
Os04
"~
Ba(CIO3)~
244
H
246
0
247
86
248
R -- H,H
249
R = 0

of 7-carbomethoxyheptana1244 with carbobenzyloxymethylidenetriphenylphosphorane 2 4 5 . The aldehyde 2 4 4 was synthesized by first oxydation of methyl
finoleate to 7-carbomethoxyheptano1243119) and then oxydation with chromium
trioxide pyridinium complex. Reaction with paraformaldehyde converts the diol
into the cyclic acetal 2 4 8 .
Hydrogenolysis of the benzylester group to the acid, reduction of the acid function
v/a the mixed ethyl carbonic anhydride with sodium borohydride led to the
alcohol 2 5 2 . Construction of the remaining side chain followed well established
procedures. The analogous 10-oxo compounds were prepared by first synthesizing
the cyclic carbonate by reaction of the dio1274 with phosgene to 2 4 9 and then
using reactions analogous to those described above. The end products consisted of
an inseparable mixture of the 150t- and 15/3-epimers.
The products 2 5 5 and 2 5 6 showed weak activity in the gerbil colon smooth muscle
contraction assay.
O
R:~O--]@\~',.,'~~O /
1) H2/Pd
O-.~**~~O/
R:::~o~OH
252
250
251
R = H,H
R~-O
0
OIt
0
254
253
0
1) Zn(BH,)2
2) hydrolysis
OH
255 R = H,H
256R
= 0
2.16. 9,11-Dihetero and 9,10,11-Triheterohomoprostanoids

In a recent publication 12~ the synthesis of a whole series of 9,11.dihetero and
9,10,11-triheteroprostanoids has been described. The common starting material
87
D. Orth and H.-E. R a d u n z
for the preparation of these compounds was oleic acid, that means that all
prostanoids synthesized had a homoprostanoid structure.
Oleic acid was converted to the erythro dio1264 or the threo diol 258 by reaction
with permanganate or hydrogenperoxide respectively. The threo compound 258
was converted to the 9,11-dioxahomoprostanoids. Reaction with paraformaldehyde
formed 259 or 260, phosgene converted the diol into 261 whereas reaction with
thiophosgene gave compound 262.
The corresponding 9,11-dioxa-10-thiahomoprostanoid 263 was synthesized by
reaction of the dio1258 with thionyl chloride. When the reaction with thionylchloride was carried out with the erythro dio1264 the isomeric 9,11-dioxa-10thiahomoprostanoid 265 with cis side chains was obtained. The 9,11-dithiahomoprostanoid 267 was prepared by reaction of the epoxide 266 with potassium methyl
xanthate.
The compounds 261 and 263 were found to be more potent than PGE 1 or PGE 2
in relaxing the pig tracheal chain. It was interesting to see that 265, the cis.isomer
of 263 was inactive in this test. Some of the compounds namely 261, 263, 266,
265 and 259 showed antidiarrheal effects when tested for inhibition of PGE2-induced diarrhea in mice.
PG-synthetase inhibitory activity was found by testing the compounds 266 and 267.
263
H
H H O ~ o 2 H
O - ~ , , " ~ ~
_
R 2 = : ( ~ L U 2 K I
f~
258
259
260
261
262
R~ = H; R2 = H,H
Rt = CHa; R2 = H,H
R1 = CHa; R2 = O
R~ = CHa; R2 = S
H
257
266
HO"~\\X~rr~
ru
267
H
/
O
'
OS~ ~[. \ x \ ~ / ~ / ~ / ~ C O
0 ~ * ~ - ~ ~
264
88
265
2C H 3

3. Conclusions
The publications which were selected for the preceeding review dealt with the
synthesis of heteroprostanoids. Patents were added only as an exception.
The amount of publications about the synthesis of heteroprostanoids proves
that the introduction of hetero atoms into the structure of prostanoid acid is
considered to be a promising starting point.
At the present time it is hardly possible to make a final judgement about the influence of such manipulations in order to promote qualitative and quantitative
changes in the biological activity. Specifications are often lacking in this matter.
The clarification of the commercial use of the analogues synthesized in the
laboratories of some firms is surely one reason for the few results reported so far.
On the other hand many of the published results can only be interpreted very carefully, because the biological models used are different.
It is remarkable, that most heteroprostanoids are very weak or no substrates for the
15-hydroxy-PG-dehydrogenase. The first metabolic step observed by naturally
occurring prostanoids apparently is stopped or slowed down already through the
introduction of a hetero atom.
There is a chance therefore to find analogues similar to the 15- or 16,16-alkylated
prostanoids of prolonged activity among the heteroprostanoids.
The strength of activity of some compounds is comparable to that of natural
prostanoids. Some heteroprostanoids also show a remarkable specifity of activity
or antiprostaglandin activity. Nevertheless one must wait for a more precise
judgement until further detailed results are published.
In the following table the heteroprostanoids and their biological activities are
summarized (see Table 1). Only those heteroprostanoids were selected which were
structurally similar to natural prostanoids.
89
'~
OH
..~w~S-~/COOH
OH
O..-OvCOOH
E-type
6H
.-~/O~COOH
OH
HO
HO
-
HO
HO
F-type
Table 1. Comparison of biological activities of some E-and F-type heteroprostanoids
No substrate for 15-hydroxy-PG-dehydrogenase (from rhesus monkey

lung), compared to PGF2~ 121,122)
No substrate for 15-hydroxy-PG-dehydtogenase from rhesus monkey

lung), compared to PGF2u121)
Not reported
Not reported
Activities
~a
e~
=
ca,
m"
OH
OH
HO
HO
HO
HO
(/~\\~ S
CO0 H
OH
~ o o H
OH
Inhibition of gastric ulcers, decrease of blood pressure 74)
4 x 10- 4 PGE 1 smooth muscle stimulating activity (gerbil colon) 123)
5% of the activity of PGFI~, (gerbil colon) 0,I x PGFla stimulation

of c-AMP (mouse ovary) Substrate for 15-hydroxy-PG-dehydrogenase
(swine lung), K m = 0,4 mM 72)
Stimulates c-AMP (mouse ovary) corpus luteum binding 1/10 PGF2a

inhibitor of 15-hydroxy-PG-dehydrogenase (from human placenta),
(/)5o = 5,2 nM 65)
,<
>
m
ca,
OH
OH
~ O O C H
E-type
~)o Table 1 (continued)
OH
H:
HO~
F-type
-OH
OH
OH
OH
Not reported
Not reported
Not repotted
Not reported
Substrate for 15-hydroxy-PG-dehydrogenase74)
Activities
er~
OH
'" ~ " ~ ' ~ C O O H
On
OH
OH
OH
OH
'~.--_I"~/~COOH
OH
..,~....,,x.,/~/COO
Not reported
Not reported
0,05 PGE 2 on s m o o t h muscle (gerbil colon) 80)
0,005 x PGE 2 on s m o o t h m u s c l e (gerbil colon) 86)
0,005 x PGE 2 on s m o o t h muscle (gerbil colon) 86)
r~
,,,r
~o
4~
E-type
Table 1
OH
HO CH3
(continued)
HO CH 3
CH 3 OH
[ ~ ' ~ C O O H
CH3 OH
~. . ~ o ~ o o H
OH
01t
OH
F-type
Stimulation of smooth muscle (rat ileum), blood pressure lowering

EDs0 = 0,2 mg/kg, inhibition of gastric ulcers (rat) 116)
Rat uterus antagonistic against PGF2~ (in vitro) 114)
Remarcable antifertility in hamsters 114)
Blood pressure lowering in cats and dogs (i. v. or orally)114)
No stimulation of rat uterus (in vitro) It4)
Activities
r-
"i
t-~
4. References
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t)
2)
3)
4)
95

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66) Fried, J., Sih, J. C.: Tetrahedxon Lett. 1973, 3899
67) Jarahak, J.: Proc. Nat. Acad. Sci. U.S. 69, 533 (1972)
68) Fried, J. Heim, S., Sunder-Plassmann, P., Etheredge, S. J., Santhanakrishnan, T. S.,
Himizu, J.: Proc. Prostaglandin Syrup.of the Worcester Foundation for Experimental
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180, 38 (1971)
70) Schenck, G. O., Dunlap, D. E.: Ang. Chem. 68, 248 (1965)
71) Sable, H. Z., Anderson, T., Talbert, B., Pasternak, Th.: Heir. Chim. Acta 46, 1157 (1963)
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74) Bruin, J. W., de Koning, H., Huisman, H. O.: Tetrahedron Lett. 1975, 4599
75) Vlattas, J., Dellavecchia, L.: Tetrahedron Lett. 1974, 4459
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(1968)
77) Vlattas, J., DeUavecchia,: Tetrahedron l_ett. 1974, 4267
78) Vlattas, J., Dellavecchia,: Tetrahedron Lett. 1974, 4455
79) Ohno, M., Narnse, N., Terasawa, J.: Otg. Synthesis 49, 27 (1969)
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81) Vlattas, J., Ong Lee, A.: Tetrahedron Lett. 1974, 4451
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87) Giantureo, M. A., Friedel, P., Giammarino, A. S.: Tetrahedron 20, 1763 (1964)
88) Zwicky, G., Waser, P. G., Eugster, C. H.: Helv. Chim. Acta 42, 1177 (1959)
96

89) Brit. Pat. 568 402 (1945)
90) Corrodi, H., Hardegger, E., K6gl, F.: Heir. Chim. Acta 40, 2454 (1957)
91) Mantione, R.: Bull. Soc. Chim. France, 1969, 4523
92) Loebich, F., Kr~imer, J. M. (E. Merck Research Lab.),: unpublished results
93) tlanessian, S., Dextraza, P., Fougerousse, A., Guidan, J.: Tetrahedron Lett. 1974, 3983
94) Lourens, G. J., Koekemoer, J. M.: Tetrahedron Lett. 1975, 3715
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96) Sowa, W.: Can. J. Chem. 46. 1586 (1968)
97) Orth, D. (E. Merck Research Lab.): unpublished results
98) Corey, E. J., Nayori, R.: Tetrahedron Lett. 1970, 311
99) Prepared by reaction of 1.2-oxidoheptane with methyl amine in EtOH under pressure
100) Corey, E. J., Weinschenker, N. M., Schaaf, T. K., Huber, W.: J. Amer. Chem. Soc. 91,
5675 (1969)
IO1) Bruhn, M., Brown, C. H., Collins, P. W., Palmer, J. R., Dajani, E. Z., Pappo, R.:
Tetrahedron Left. 1976, 235
102) Collins, P. W., Pappo, R.: Prostaglandins Vol. 10, No. 5,733 (1975)
103) Heslinga, L., van Gorkem, M., van Dorp, D. A.: Rec. Tray. Chim. Phys.-Bas 87, 1421 (1968)
104) Heather, J. B., Sood, R., Price, P., Pernzzotti, G. P., Lee, S. S., Lee, L. F. H., Sih, C. J.:
Tetrahedron Lett. 1973, 2313
105) Radunz, H. E., Kr/imer, J. M. (E. Merck Research Lab.): unpublished results
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107) Corey, E. J., Chaykovsky, M.: J. Amer. Chem. Soc. 87, 1353 (1965)
108) Umbach, W., Mehren, R., Stein, W.: Fette, Seifen-, Anstrichmittel 71,199 (1969)
109) Miyano, M., Mueller, R. A., Dorn, C. R.: lntra-Science Chem. Rept. Vol. 6, No. 1, 43
(1972)
110) Miyano, M., Stealey, M. A.: J. Org. Chem. 40, 1748 (1975)
111) Miyano, M., Dorn, C. R., Mueller, R. A.: J. Org. Chem. 37, 1810 (1972)
112) Diinnschichtsehromatographie Stahl, E. (Ed.), Springer-Verlag, Berlin, Heidelberg,
2. Auflg., S. 631. New York: 1967
113) Pappo, R., Collins, P., Jung, C.: Tetrahedton Lett. 1973, 943
114) We thank Dr. Schliep, Dr. Hatting (E. Merck, Medical Research Laboratories) for performing the biological tests
115) Orth, D., (E. Merck, Research Lab.) unpublished results
116) DOS 2323193, (E. J. Du Pont de Nemours and Co., USA)
117) Harrison, J. T., Fletcher, V. R.: Tetrahedron Lett. 1974, 2729
118) Harrison, J. T.o Grayshan, R., Williams, T., Semenovski, A., Fried, J. H.: Tetrahedron
Lett. 1972. 5151
119) Sehauenstein, E., Esterbauer: Fette, Seifen, Ansttichmittel, 70, 4 (1968)
120) Bender, A. P.: J. Med. Chem. 18, 1094 (1975)
121) Sun, F. F., Armour, S. B., Bockstanz, V. R., McGuire, J. C.: Adv. in Prostaglandin and
Thromboxane Research (Samuelsson, B., Paoletti, R., eds.), Vol. 1, p. 177 New York:
Ravens Press
122) Germ. OS 2423156 (Upjolm Company, USA)
123) Fried, J., Mehra, M. M., Kao, W. L., Lin, C. H.: Tetrahedron Lett. 1970, 2695
124) CrabbE, P., Cervantes, A., Meana, M. C.: J. Chem. Soc. Chem. Comm. 1973, 119
125) Crabb~, P., Guzman, A., Velarde, E.: J. Chem. Soc. Chem. Comm. 1972, 1126
126) Gandolfi, C., Doria, G., Pharmaco, I1: Ed. So. 29, 327 (1974)
12"/) Dikshit, D. K., Kapil, R. S., Anand, N.: Indian J. Chem. 13, 1353, 1359 (1975)
Received June 18, 1976
97
Dr. Erich Schacht

Pharmaceutical Research Department, E. Merck, 6100 Darmstadt, Germany
Table o f C o n t e n t s
A. I n t r o d u c t i o n
100
B. S u b s t i t u t e d A r y l o x y a c e t i e Acids
. . . . . . . . . . . . . .
1. a - A r y l o x y i s o b u t y r i c Acids
. . . . . . . . . . . . . . .
2. 2-Phenoxyalkylic Acid Derivatives . . . . . . . . . . . . .
3. 2 - P h e n o x y p h e n y l a c e t i c A c i d Derivatives . . . . . . . . . . .
4. Bisaryloxyacetic A c i d Derivatives . . . . . . . . . . . . .
5. Miscellaneous Acids . . . . . . . . . . . . . . . . . .
101
102
103
103
104
104
C. C h e m i s t r y
105
D. H y p o l i p i d a e m i c A c t i v i t y . . . . . . . . . . . . . . . . .
1. M e t h o d s . . . . . . . . . . . . . . . . . . . . . .
2. Results . . . . . . . . . . . . . . . . . . . . . .
E. Discussion . . . . . . . . . . . . . . . . . . .
1. 0~.Aryloxyisobutyric Acid Derivatives . . . . . . . .
2. ,v-Aryloxypropionic Acid Derivatives . . . . . . . .
3. t~-Aryloxyphenylacetic A c i d Derivatives . . . . . . .
4. r , - A r y l o x y h y d r a t r o p i c A c i d Derivatives
. . . . . . .
5. Bisaryloxyacetic A c i d Derivatives . . . . . . . . .
6. A r y l o x y a l k a n o l Derivatives . . . . . . . . . . .
F. R e f e r e n c e s .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
. . . .
.
117
117
118
118
.
.
.
.
.
118
118
119
119
119
120
121
E. Sehacht
A. I n t r o d u c t i o n
The increase of plasma lipids, formerly called hyperlipidaemia, in addition to
hypertension, overweight, nicotine abuse, diabetes mellitus and certain environmental effects belongs to the so-called risk factors of arteriosclerosis. It was discovered that the water insoluble lipids (cholesterol, triglycerides and phospholipids)
are transported in plasma by forming a macromolecular complex with specific
proteins; this disorder is nowaday usually called hyperlipoproteinaemia, which today
represents the most frequent metabolic disease. After it was found that cholesterol
is the main lipid component of the arteriosclerotic lesions in the vascular wall,
the number of investigations dealing with the correlation between plasma cholesterol
concentration and the development of arteriosclerosis increased. The coincidence
of hyperlipoproteinaemias and coronary heart disease may be considered established
on the basis of comprehensive retrospective and prospective studies I - 5 ). In a study
by Carlson e t al. 1 ) it was recently demonstrated that in males up to 60 years an
elevated triglyceride serum level, independent of cholesterol, significantly increases
the incidence of coronary diseases. The therapy of hyperlipoproteinaemia is an
important goal in preventive medicine. Various primary preventive studies have indeed shown that the normalization of pathological values reduces or delays the risk
of dying from the complications of arteriosclerosis. Hyperlipoproteinaemia is now,
furthermore, recognized to be at least one of the factors involved in the pathogenesis
of several other diseases.
Arteriosclerosis
Hyperuricaemia
Hyperinsulinaemia
Gallstones
Hyperli fidaemia
~" Pancreatitis
Skin Lesions
Decreased Glucose
Tolerance
Fatty Liver
This is an adequate basis for a therapy, although the biochemical pathogenetic
correlations between increased blood lipids and arteriosclerosis or other diseases
are largely unknown. In any case a lipid lowering therapy will be useful only if
started early and understood as long-term therapy.
I00
HypolipidaemicAryloxyaceticAcids
B. S u b s t i t u t e d A r y l o x y a c e t i c Acids
Clofibrate: Clofibrate [ethyl-2-(p-chlorophenoxy).2.methyl-propionate], 1, is one
of the more frequently prescribed drugs in current use for the management of
hyperlipoproteinaemia of various origins. Although the ethyl ester of this acid
has been reported by Julia et aL 6), it was Thorp and Waring 7) who discovered that
this compound and the free acid possess favorable hypolipidaemic activity in the
rat. Clofibrate has been shown to be an effective lipid lowering drug in man 8' 9)
It is more effective in lowering triglycerides than serum cholesterol 7' 11). The drug
rapidly undergoes hydrolysis in vivo, and the corresponding acid is presumed to
be the active drug l~ It seems that clofibrate may be exerting its effect by multiple
modes of actions 12). Included among the proposed mechanisms of action are a
decrease in the synthesis 13-1S)and an increase in the catabolism of cholesterol and
low density lipoproteins in the liver, decrease in hepatic lipoprotein secretion s6),
decrease in plasma unesterified fatty acid concentrationlT' 1s), increase in the rate
of conversion of cholesterol to bile acids in the liver 27), decrease in the rates of
synthesis 19) and secretion of triglycerides in the liver and increase of the breakdown
of triglycerides and very low density lipoproteins in the peripheral tissues and
alterations in thyroid hormone distribution 2~ 21). The hope raised by three more
comprehensive investigations2~ ~4) that clofibrate independent of its effect on
raised blood fats might possess still other infarction-preventing properties does
not seem to be confirmed 2s). The results of the Coronary Drug Project 2s) demonstrate that primary prevention is far superior to secondary prevention, because
the prognostic importance of lipid levels is much less in patients after cardiac
infarction than before the infarction. The highest risk of infarction is a previous
infarction 26). In addition, the latter study 2s) demonstrated only a slight lowering
of the cholesterol level by 6.5% under clofibrate medication, while the triglycerides
showed a better response with 22.2%. The necessary search for a more potent
compound appears confirmed by this study.
CloJ~brate derivatives: Several direct derivatives of clofibrate with comparable
activities are commercialized or in the last stage of clinical trial, e. g. alufibrate as
Atherolip (R) 28' 29)(hydroxy-aluminium bis-[2-(p-chlorophenoxy)-2-methylpropionate]), clofibride or MG 46 as Lipenan (R) (4-hydroxy-N-dimethylbutyramide4-chloro-phenoxy-isobutyrate)3~ or simfibrate or CLY-503 (1,3-propanediol-bis[2-p-chlorophenoxy-isobutyrate])31 ).
Compounds combining the structural elements of both the proven lipid
lowering drugs clofibrate and nicotinic acid or of the corresponding alcohol
~-pyridylcarbinol (Ronicol) are more interesting because they are more effective
than pure clofibrate. In this context the following products, already on the market,
should be mentioned: Etofibrate as Lipo-Merz (R) [2-(p-chloro-phenoxy)-2.methylpropionic acid-[2-(nicotinoyl-oxy)-ethyl ester] 32' 33) and clofenpyride or ATE
as Arterium-V (R) [3-hydroxy-methylpyridine-(p-chlorophenoxy)-~-isobutyrate
hydrochloride] 34). The potentiation of the lipid lowering activity of clofibrate
by combination with low doses of 3-pyridylcarbinol was discovered by Simane and
Nowak 3s). A combination of clofibrate plus ~pyridylcarbinol 20: 1 (Liapten (R))
was selected, and clinical trials confirmed the synergistic acitivity36).
101
E. Schacht
1. a-Aryloxyisobutyrie Acids
A larger number of aryloxyisobutyrie acids have been prepared for hypolipidaemic
screening. The reason for this was the great success of clofibrate, but it might also
be the ready availability of these compounds. Phenols react readily in acetone
with chloroform in the presence of a strong base to furnish a-substituted isobutyric
acids 37)-. Among these investigational drugs only a few were of some importance,
but none obtained the importance of clofibrate. A number of clinical publications
on nafenopine or SU-13,437 [2-methyl-2-[(p-1.2.3.4.-tetrahydro-l-naphthyl)phenoxy] propionic acid] appeared during recent years 38-42). In general, results
showed greater reduction of serum triglycerides than of serum cholesterol. Early
claims for an advantage over clofibrate in treatment of type II hyperlipidaemia have
not been confirmed. In a comparative study of the two drugs, Dujovne et al. found
nafenopine (600 mg per day) slightly, but not significantly superior to clofibrate
(2 g per day) in overall reduction of serum lipids, but cholesterol reduction was not
superior in type II, and there may have been an "escape" of serum triglycerides
in the type IV patients with nafenopine 38). The detection of liver pathology in
long-term, high-dosage studies in rats has resulted in withdrawal of nafenopine from
further clinical trials4~ Methylclofenapate or ICI 55,695 (methyl-2-[4-(p-chlorophenyl)-phenoxy]-2-methyl-propionate), a biphenyl derivative of the methyl ester
of clofibrate, was slightly more effective than clofibrate in reducing cholesterol in
type II patients, and it was much more effective than clofibrate in reducing
cholesterol and triglycerides in type III and IV patients 43). The compound was
withdrawn after preliminary trial in humans because it was found to have late
hepatotoxic properties in mice and rats 43). The effect of S-8527 3 (I.1 .-bis-[4'(l"-carboxy- l"-methyl-propoxy)-phenyl] cyclohexane) on cholesterol metabolism
and serum and liver lipids in rats has been studied 44- 47). S-8527 has been reported to possess pronounced hypolipidaemic properties in experimental animals and
is considered to be more potent in hypolipidaemic activity and less potent in hepatoCH3
CI-O--O--{--COO--C2H s
CH3
[1 ]
CIofibrate
/ \
--
2Hs
CH3
~
CH3
/~--O--~---COOH"
C2H5
13]
S-8527
CH3
I
O--C--COOH
I
CH3
[2] Su-13,437, Melipan,Nafenopine
102
N---J 6-~
[4]
--x:/
AT-308
I
CH3
megalic effect than clofibrate 4s' 46). In pharmacological tests on hypercholesterolaemic or normocholesterolaemic rats AT 308 4 (3-[4-( 1-ethoxycarbonyl- 1-methylethoxy).phenyl]-5-(3-pyridyl)-1.2.4-oxadiazole) showed the highest hypocholesterolaemic activity amongst several compounds of a large series 48). The higher activity
of ethyl.2-(dibenzo.furanyl-4-oxy)-2-methyl-propionate against clofibrate in mouse
and rat models is reported by a Swedish research group 49' so). Another new cx-aryloxyisobutyric acid BM 15.075 (2-[4-chloro-benzamidoethyl)-phenoxy]-2-methylpropionic acid) was recently shown to be about 20 times as potent as clofibric acid
in rat hypercholesteraemia and hypertriglyceridaemiasl). Clinical trials have been
started with this compound. In Helsinkis2a' b) some pharmacological, toxicological
and clinical results of another structural analog of clofibrate, called LF 178 or
Lipanthyl Osopropyl-2-[4.(4-chlorobenzoyl)-phenoxy]-2-methyl-propionate, were
reported. Depending on the model, Lipanthyl is 6-10 times more active than
clofibrate in animal experiments. Since the compound was nontoxic and free of side
effects, it has been commercialized.
2. 2-Phenoxyalkylic Acid Derivatives
Among a number of 2-phenoxyalkylic acids, which were synthesized and tested s3),
only two compounds have obtained greater importance. The first compound,
GP-45699 [D,L-2-(p-diphenyloxy)-heptanoic acid] has been prepared by Nardi
et al. s4), and has been tested in man ss' s6). The compound gave a greater and more
sustained reduction of plasma cholesterol than clofibrate, but the side effects were
too common and severe to justify its routine use ss). The second one, HCG-004 or
fenofibric acid 5 (2-[4-(4'-chlorophenoxy)-phenoxy]-propionic acid), has been
synthesized and tested by a German research group sT). In normolipidaemic and
hyperlipidaemic animals HCG-004 was a well-tolerated and highly effective oral
hypolipidaemic drug. Within the hypolipidaemically interesting dosage range no
other pharmacological or chronic-toxicological effects were found s7, sa) Therefore, clinical trials have been started, but no recent results are available.
3, 2-Phenoxyphenyla~cetic Acid Derivatives
Halofenate or MK-185 6 [2-acetamidoethyl-(p-chlorophenyl)-(m-trifluoromethylphenoxy)-acetate] is the best known compound of the 2-phenoxy-phenylacetic
acid type. Its hypolipidaemie activity has been proved by several research groups s9-64).
In rats, halofenate reduced both cholesterol and triglycerides, with a potency 5.7
times that of clofibrate, but in man the changes of cholesterolaemia were minimal,
inconsistent and not statistically significant. Halofenate was found, on the contrary,
quite effective compared to clofibrate in reducing plasma triglyceride levels.
Halofenate is the only drug among the aryloxyacetic acids which has been found to
induce a very notable decrease of uricaemias6, 63) This may indicate a special
rationale for this drug in the not uncommon type IV patient with mild diabetes
and hyperuricaemia64). In the meantime halofenate is commercialized under the
trade name Livipas (R) in Great Britain.
103
E. Schacht
4. Bisaryloxyacetic Acid Derivatives
In this structural series Sail 42-348 or lifibrate 7 [ 1-methyl-4-piperidyl-bis(pchlorophenoxy)-acetate] is the best studied compound. It has been under investigation for the past 8 years. A lot of publications can be found in the literature
6s-71). Sail 42-348 was reported to be an effective hypolipidaemic agent, 9 times
more potent than clofibrate in male rats 6s). Clinical trials have demonstrated its
efficacy in the treatment of type II hyperlipoproteinaemic disorders 66). However,
recent observations in one of 4 patients treated with low doses of Sail 42-348 in
a pilot study suggested acute hepatotoxicity 69). It must be supposed that Sail
42-348 has been withdrawn from further clinical trials. Three more, direct derivatives
of Sail 42-348, have been described in the literature, but they are not of higher
importance than the parent compound st' s2a, c, 72). Of more interest in this
structural class is treloxinate 8 (methyl-2,10-dichloro-12 H-dibenzo[d, g], [ 1, 3]dioxocin.6.carboxylate)73, 74). The p-chlorophenyl substituents are held in a
O-o
CH--COO~N--CHs
CHa
[5]
HCG-004,Fenofibricacid
o_~o /
[7]
x__/
Sail 42-348, Lifibrate
cI
Clio
O-CH-COO-CH 2-CH2-NH-C-CH 3
CI/~
CF3
[6]
H2C./'"
MK-185,Halofenate,Livipas
[8]
~CH--COO--CHa
O
Treloxinate
fixed conformation as part of the dioxocin ring structure. In rats treloxinate is

8 times as potent as clofibrate in reducing plasma cholesterol, and 30 times
as potent in reducing triglycerides. A large number of derivatives of treloxinate
have been synthesized and tested for hypolipidaemie activity in rats 74). The drug
is currently undergoing clinical trials, but no recent results could be found in the
literature.
5. Miscellaneous Acids
The discovery of the hypocholesterolaemic properties of probucol or DL-581
[4.4-(isopropylidene-dithio)bis(2.6-di-t-butyl-phenol)] led to a structure-activity
study among related compounds 7s). The result of this study was the selection of DL
104
990 [2-(3.5-di-t-butyl-4-hydroxy-phenylthio)-hexanoic acid] for further development.
DL-990 possessed better hypoeholesterolaemie and especially more potent hypotriglyceridaemic properties than probucol sl). The clinical examination has been
initiated. ICI 59 897 [2-(4'-chloro-biphenylyl-(4)-methoxy)-2-methyl-propionic
acid] must be considered as a drug with slighter hypolipidaemic activity, but with
more pronounced beneficial side effectssl). RMI 14,514 [(5-tetradecyl-oxy)-2furoic acid] is more effective than clofibrate in reducing plasma cholesterol levels.
Plasma triglycerides are reduced to approximately the same degree by both agents76).
The hepatomegaly was significantly less than that seen after treatment with clofibrate.
The synthesis of a new potent antihypercholesterolaemic agent, Wy-14643
[4-chloro-6-(2.3-xylidino)-(2-pyrimidinylthio)-acetic acid], has recently been reported 77). The activity of this drug depends very much on the animal models used.
Wy-14643 had only a slight effect on the serum cholesterol of normal rats, but the
compound proved to be more potent than clofibrate by a factor of about 100 in
the hypercholesterolaemic rat model sx' 78). Tibric acid, CP-18.524 or Exirel (R)
[2-chloro.5-(cis-3.5-dimethylpiperidino-sulfonyl)benzoic acid] is a member of a new
class of drugs which produces hypolipidaemic effects in rats sl' 79). The effects of
tibric acid on hyperlipoproteinaemia in man were studied in several different
hospitalsSl, so-82). This novel, generally well tolerated drug is more useful in the
treatment of hypertriglyceridaemias than of hypercholesterolaemias. Real advantages
over clofibrate could not be noted. The compound was recently commercialized in
Switzerland.
It can be seen that several drugs mentioned are useful lipid lowering agents.
Generally, however, no drug is known which completely normalizes hyperlipidaemia.
Consequently, a search for drugs which are equipotent in affecting the hypercholesterolaemia as well as hypertriglyceridaemia was made in this laboratory.
Furthermore, it is necessary to seek for lipid lowering agents which are more specifically effective against various types of hyperlipoproteinaemias. The success of
elofibrate derivatives and their acceptance by the medical profession instigated us
to synthesize structurally similar compounds.
C. C h e m i s t r y
In the present article the syntheses of several substituted aryloxyacetic acids 9
are discussed in short concentrated form. In a later communication the detailed
preparative procedures will be reported. The clofibrate structure 1 was modified
in the carboxylic acid part by esterification with selected alcohols or by amidation
R2
I
R3~--C--COOH
RI
[91
105
E. Schacht
with special amines or by reduction to the corresponding alcohols. One or both
of the two methyl groups at the a-carbon atom were replaced by hydrogen, aryl
or aryloxy groups. In several cases the p-chlorine.atom of the clofibrate structure
was substituted by aryl, aryloxy and aryloxymethyl groups, isocyclic and heterocyclic ring systems. The choice of the moieties was not done arbitrarily, but the
well-known above mentioned lipid lowering agents formed a structural framework.
You will find direct clofibrate derivatives, substituted with heterocyclic groups
22-24, isocyclic and heterocyclic nafenopine compounds 25, 26, 31-34, piperidyl
substituted methylclofenapate analogs 27-30, with different heterocyclic moieties
substituted halofenate structures 42-46 and substituted lifibrate compounds of
various structures 54-5Z Furthermore, a direct structural alteration of the
fenofibric acid was performed with the aryloxymethyl types 39-41. A selection
of the prepared compounds will be found in Tables 1-6.
Table 1. a-Aryloxyisobutyricacid derivatives
CH3
CH3
Cmpd.
R3
R4
mp. ~
22
~N--
-OH
105-107
23
~N--
-OH
203-205
24
~N--
-OH
159-160
25
~ - -
-OH
202
26
-OH
125-127
27
~ N - ~
-OH
224-226
-OOH
106
Hypolipidaemir AryloxyaceticAcids
Table I (continued)
Compd.
R3
R4
mp. ~ 1)
29
C N ' - ~
-NH 2
210-212
31
H--N~
-OH
222-2242 )
32
H3C--~
-OH
221-2232)
33
~/---~q--
-OH
115-117
198-2012 )
34
~/-~
-OH
220-2252 )
137-1393 )
35
CI--~-O--CH
2-
-OH
153
36
Br~-O--CH
2-
-OH
170-172
37
F-,~O--CH2--
-OH
123-125
1) Uncorrected.
2) Cyclohexylaminesalt.
3) Diisopropylaminesalt.
107
E. Schacht
Table 2. wAryloxypropionicacid derivatives88, 91, 92)
He,
I II
R3t - - - - ~ - ~ - C R4
CHa
ff--~
1) Compd. R3
R4
mp. ~
38
HaC--N~)---
-OH
203-2052)
39
CI-~O--CH2--
-OH
158-159
40
CI--~O--CH
2-
-OCH3
70-71
41
F ~ --~- k-=-/ - C H-
2-
-OH
143- 144
1) Uncorrected.
Table 3. a-Aryioxyphenylaceticacid derivatives88, 89, 92)

H O
R3-~O~[CI
- R4
Rs
Compd.
42
43
44
108
R3
~N m
H3C--~~)'--
~--
R4
R5
rap. ~ 1) (nD20)
-OH
p-Cl
173-175
-OH
165-1682 )
-OH
169-1712 )
Table 3 (continued)
Compd.
E3
45
46
R4
R5
-OC2H5
p-Cl
-OH
mp. ~ 1) (riD20)
(1,6129)
239-2413)
1) Uncorrected.
3) Cyelohexylaminesalt.
Table 4. ~-Aryloxyhydratropicacid derivatives88, 89, 93)
CHs
Compd.
47
R3
C1--
R4
mp. ~ 1)
-OH
101-102
48
C I ~
-OH
127
49
H3C--~
-OH
172; 190-1922)
-OH
178-1802 )
93-96
50
O=~N--
51
-OC2H5
52
S/-'-~
-OH
187-1892 )
109
E. Schacht
Table 4 (continued)
Compd.
53
R3
~
R4
mp. ~
-OH
195-1972 )
1) Uncorrected.
Table 5. Bisaryloxyaceticacid derivatives95, 96)

R3--~O~
CH-COO-Rs
R,--~O /
Compd. R 3
R4
-
CN
mp. ~ 1)
-C2H 5
2082)
122-123
54
CN
55
C1-
Cl-~O--
-H
56
CI-
CI--~O--
_ C2H5
57
C1-
HaC-N/"'~
O
1) Uncorrected.
2) HCI salt.
110
R5
-H
(nD20)
(1,5796)
152-1553 )
HypolipidaemicAryloxyacetic Acids
Table 6. Aryloxyalkanol derivatives97)
R4
R3--~O-~-CHz-O-R6
Rs
Compd. R 3
R4
Rs
R6
mp. ~
58
CN__ ~
-CH3
-CH3
-H
158-160
59
C1-~O--
-H
-CH3
-H
30-32
60
CI
-H
-CH 3
O
-C-CH 3
61
CI--~O--CH
2-
-H
-CH 3
-H
77-78
62
CI--~---CH
2-
-CH 3
-CH 3
-H
67-70
63
F-~O--CH2--
-CH 3
-CH 3
-H
108-109
64
H3C--]q'/~/--
-CH 3
-CH 3
- H
92-94
O--
(nD20)
(1,5550)
1) Uncorrected.
The compounds of structure 9 were obtained by condensing a phenol of
formula 10 with a compound of formula 11 and following saponification or in
the special case of R 1 = R~ = CH 3 with chloroform and acetone in the presence
of potassium hydroxide 37J. The compounds 11 were commercially available or
readily prepared by ~x-halogenation of the corresponding esters 12.
R3-~OH
[]0]
Hal-C-CO 2C 2 Hs
H - C - C O 2 C2 H5
R2
11
Rl
12
111
E. Schaeht
The other starting materials, the phenols 10, were only in part commercially
available. Some of these compounds were synthesized by methods described in
the literature, but a greater part of them were unknown and new procedures had
to be developped for them.
In the course of our synthetic program on hypolipidaemic aryloxyacetic aids
all the following phenols have been prepared.
4-(4-Chlorophenyl)-phenol was obtained by the method of Savoy and
Abernethy as), 4-(4-chlorophenoxy)-phenol by the procedure of Granzer and Nahm sT)
and 4-(1.2.3.4-tetrahydro-l.naphthyl)-phenol by the route used by Hess and Bencze
86)
4-Piperidinophenol 13 was prepared by two different ways [Eqs. (1) and (2)]:
first by reacting p-anisidine with 1.5-dibromopentane and treating the resulting
4-piperidino-anisole with HBr, and second by the homolytic aromatic amination of
phenol with N-chloropiperioine
9 83) .
4-Pyrrolidino-phenol was formed analogously from p-anisidine and 1.4dibromobutane. 4-Isoindolino-phenol was obtained by reacting p-anisidine with
o-xylilene dibromide to form 2-p-methoxyphenyl-isoindoline, and splitting the
ether with HBr.
4-(1-Pyrryl)-phenol 14 was obtainable by reacting p.amino-phenol with 2.5-dimethoxytetrahydrofuran [Eq. (3)]. 4-(4-piperidinophenyl)-phenol could be easily
prepared in two different ways. One of the synthetic possibilities was the reaction
of 4-amino-4'-methoxy-bipheny184' 94) with 1,5-dibromopentane to form 4-piperidino-4'-methoxy-biphenyl 1.5 and splitting the ether with HBr. The other
possibility to get 15 and the corresponding phenol was the nucleophilie amination
of 4-iodo-4'-methoxy-bipheny198) with excess piperidine. Starting material for both
procedures was 4-phenylphenyl-benzoate [Eq. (4)].
4-(2-lndanyl)-phenol 16 was obtained by reacting p-methoxy-phenyl-acetic acid
ethyl ester with benzylchloride to form a-benzyl-p-methoxyphenyl ethyl acetate,
saponification into the acid, conversion of the acid with thionylchloride into the
chloride, cyclization to 2-p-methoxy-phenyl-l-indanone, NaBH4 reduction to 2-pmethoxyphenyl-l-indanole, dehydration with p-toluene-sulphonic acid in toluene
to 2-p-methoxyphenyl-indene, catalytic hydrogenation to 2-p-methoxyphenylindene, and treating the ether with HBr [Eq. (5)].
For the preparation of 4-(l.2.3.4-tetrahydroquinolino)-phenol 1 7 three
different procedures were worked out [Eqs. (6)-(8)]. 17 was prepared by reacting
N-p-methoxyphenyl-anthranilic acid with acetic anhydride and subsequent saponification to 1-p-methoxyphenyl-4-hydroxy-2-quinolone, reaction with POCI3 to
form l-p-methoxyphenyl-4-chloro-2-quinolone, hydrogenation to 1-(p-methoxyphenyl)-3.4-dihydro-2-quinolone, splitting the ether with HBr to 1-(p-hydroxyphenyl)3.4-dihydro-2-quinolone, and reduction with LiA1H4 [Eq. (6)]. Another
synthetic possibility was the reaction of p-anilinophenol with ~-propiolactone and
subsequent cyclization to l-(p-acetoxyphenyl)2.3-dihydro-4-quinolone 18. The
next step, the Wolff-Kishner reduction, led directly to the desired product [Eq. (7)].
The third way, the direct amination of p-iodoanisole with 1.2.3.4-tetrahydroquinoline and the subsequent splitting of 4-(1.2.3.4-tetrahydro-quinolino)-anisol
with HBr was the best one [Eq. (8)]. Saponification of 1-(p-acetoxyphenyl)-2.3112
HypoUpidaemicAryloxyaceticAcids
(1)
I HBr
(2)
[13]
H3CO-~OCHa + H2N--~OH
HOAc C N _ ~ O H
(3)
[14]
O2N~ ~ ' - O H
(CH3)2SO4
~-O 2 N~~ O C H 3
I KOH
0
H2
~--- H 2 N ~ ~ ~ O C H
Br-(CH2)s-BrIl K2CO3
[15]
I HNOa/H2SO4
O
H-
Cul/Cu
KzCO3
12
113
(4)
E. Schacht
'IsCO--~H2-CO2C2Hs +CI-CH2--~
DMSO/NaH
~ HsCO~~H-COzCzHs
2
KOH
SOCI2.~_ H 3 C O _ _ ~ H _ C O C I
CHz
OH
OCH3
H3C~-SO3H
HBr
( 5)
=.~
CH3
H2
~~-OH
[161
dihydro-4-quinolone 18 to 1-(p-hydroxyphenyl-2.3-dihydro-4.quinolone was carried

out with potassium hydroxide in ethanol. 4-(1.2.3.4-Tetrahydro-4-quinolyl)phenol 19 was prepared by cyclization of p-methoxy-cinnamic acid anilide with
polyphosphoric acid to form 4-(p-methoxyphenyl)-l.2.3.4.-tetrahydro-2-quinolone,
reduction with LiAIH4 to 4-(p-methoxyphenyl-l.2.3.4-tetrahydro.quinoline 20,
and splitting the ether with HBr [Eq. (9)]. Catalytic reductive methylation of 20
with formaldehyde (H 2) 5% Pd-C gave l-methyl-4-p-methoxyphenyl-1.2.3.4-tetrahydro-quinoline. The corresponding phenol, 4-(l-methyl-l.2.3.4-tetrahydro.4quinolyl)-phenol was obtained by ether splitting with HBr. The attempts to prepare
4-p-hydroxyphenylchromane 21 and 4-p-hydroxyphenyl-thiochromane by an
analogous Friedel-Crafts-procedure were successful. 4-Chromanol or 4-thiochromanol
reacted with phenol in the presence of AIC1a to form the above mentioned products
[Eq. (10)].
114
OH
Nit
AcOAc
OCtt3
H2
CI
O
OCtt3
OCIt3
HBr- ~ N - N ~
OCH~
(6)
O LiAIH~ ~ ' - ~
OH
OH
1171
---
OH
CHz---CH2-COOH
H2C------O
(7)
HOAc~ ~
AcOAc
N2H4/KOH _-HO-CHz-CH2-OH
O-C-CHs
OH
[18]
+ I~-'OCH3
H
K2CO3/HMPT~
CuC1/Cu
(8)
OCH3
OH
115
E. Sehacht
H
O
II
HaCO--~CH=CH-C-NH--~
(9)
PPA.---
OCH3
H
OCHa
OH
[201
[19]
AICI3
+ ~---OH
(10)
OH
OH
[211
For our structural group (R 1 = H, CHa; R 2 = CHa; R 3 = R 4 - C 6 H 4 - O - C H 2 - ) ,
see formula 9, special procedures were found to be superior to the normal phenol
alkylation as the last step [Eqs. (11) and (12)].
(11)
R, = H.F,CI,Br)
H.~C_ _ ~
CH3
OH + Br-C-CO2C2Hs
Na/C2HsOH
~ ~-~
I
~ HaC
CHs
I-CO2C2Hs NBS
C C]4~-~
CH3
CH3
R4-~OH
Br CH2 - - ~ O - ~ - C O ~C~Hs
CH3
CHa
KOH
]
.-- R 4 - - ~ - C H 2 - - ~ _ V _ C O O H
CHa
Scheme I
116
CH3
~H 3
-
CHa
R,= H,F,CI,Br,)
O = C H - - ~ - - O H + Br-CH-CO2C2Hs
I
CHa
HO-CHr
NaBH4
DMF= O=CH
O- H-CO2C2Hs
CH3
SOCI2
/ \~'~O-CH-CO2C2Hs" ~ - ~ " C I - C H 2
" ~k~/
]
HMPT
CH~
O-CH-CO:CaHs
/
CH3
:- R
Scheme 2
O-CH2
(12)
Na/C2HsOH
Cyo
- H-COOH
CH3
CH3
D. H y p o l i p i d a e m i c A c t i v i t y
The investigation of the hypolipidaemic activity was done by Z. Simane at the
Biochemical Research Department of E. Merck, Darmstadt.
1. Methods
Male Wistar rats initially weighing 160-200g were used for the experiments. All
animals were kept in macrolon cages in groups of 5, weighed twice weekly and
given food (Altromin-R (R)) and water ad libitum. The compounds to be tested
were administered orally, suspended in a medium as described by Dorfman et al. 99)
(0,5 ml per 100 g body weight). The rats of the control groups received the same
volume of medium without drugs. The experiments were carried out as follows:
Rats fed basal diet
The drugs were administered for 11 days. Two hours after treatment on the 10th
day blood samples were taken retroorbitally 1~176
and analyzed for total cholesterol
lot). Then the animals received 10% fructose solution in place of drinking water
for the next 24 h. After the last administration of the test compound or mixture
the rats were killed, blood samples were taken, and the serum obtained was analyzed
for triglycerides ~o2).
PTU-Rats
The animals received drinking water containing 0,01% 6-propylthiouracil (PTU)

ad libitum. After 2 days oral administration of drugs to be tested began and was
117
E. Schacht
continued for 11 days. Blood samples for cholesterol estimation were obtained
and further treatment of the rats was carried out in the same manner as described
for rats fed basal diet. The data obtained in these experiments were statistically
evaluated by the t-test according to the method of Dunnett 1~
2. Results
The results of the screening for hypolipidaemic activity of all the synthesized compounds will be presented in a later communication. But the most striking results
will be reported in the following discussion.
E. Discussion
1. a-Aryloxyisobutyrie Acid Derivatives
Those compounds directly related to clofibrate showed more or less pronounced
effects on the lipid levels in experimental animals. Surprisingly, the substitution
of the 4-chlorine atom by the isocyclic 2-indanyl moiety 26 or by heterocyclic
moieties, for instance pyrrolidiny122, piperidyl 23, pyrrolyl 24 or isoindoly125,
largely maintained the effect. On the other hand, an increase of the lipid lowering
activities up to the factor 10 was found if the heterocyclic substitutents were
1.2.3.4.-tetrahydro-4-quinolyl 31, l-methyl-1.2.3.4-tetrahydro-4-quinolyl 32 or
1.2.3.4-tetrahydro-t-quinolyl 33. An increase of the lipophilia of the compounds
by the introduction of a 4-piperidino-phenyl moiety instead of the 4-chlorine atom
into clofibrate elevates the activity additionally. The prototype of these 4'-piperidinobiphenyl-(4)-compounds, the free acid 27, was I0 to 20 times more active
than clofibrate, depending on the experimental model. A number of compounds
derived from 27 was made by esterification or amidation of the free carboxylic
group to investigate the structure-activity relationship. The amide 29, the amino
ester 30 and the 1-glyceryl ester 28 were the most active compounds among the
investigated so-called "pro-drugs", especially with regard to their hypotriglyceridaemic
effects. Similar results were obtained in other structural classes where the carbocyclic
group was modified.
A completely different modification of the clofibrate structure was obtained by
synthesis of the phenoxy-methylphenoxyisobutyric acids. The 4'-bromo 36 and
the 4'-chloro compound 35 were found to be considerably more active than the
reference compound clofibrate. But whether definite advantages of these compounds
over clofibrate can be demonstrated will be shown only by the current investigations
on the mode of action, of side effects and toxicity.
2. a-Aryloxypropinnie Acid Derivatives
One methyl group can be substituted at the wcarbonatom of the clofibrate
structure with an hydrogen atom without loss of activity. This could be demonstrated with the identical activity of the compounds 32 and 38.
118
Similarly, as in the class mentioned above, the phenoxymethyl derivatives
of aryloxy propionic acid derivatives were found to be highly effective. Thus
compound 39 on comparing the dose response curves were 23 times more active
than clofibrate in lowering the cholesterol level and 48 times more active in
lowering the triglyceride level. In comparison to HCG-004 5, the direct structural
analog, an equal activity in lowering serum lipid levels of rats was found. Unfortunately, compound 39 in acute toxiocological experiments showed only a
small safety margin so that development was discontinued. The fluorine derivative
41 showed about the same hypolipaemic effects; its toxicological properties are
as yet unknown.
3. wAryloxyphenylacetic Acid Derivatives

Investigation of this substance class showed that both methyl groups at the a-C
atom of the clofibrate structure may be substituted by the hydrogen atom or the
phenyl group without causing a substantial loss of activity. The activity of 42
was directly comparable to that ofhalofenate 6, and compounds 43, 45 and 46
showed the same or a slightly better activity than the corresponding a-aryloxy
isobutyric acid derivatives of Table 1. Summarizing, the results are approximately
comparable to those described under Section 1.
Compound 43 which was of particular interest, showed a marked inhibition of drug
metabolizing enzymes in the liver. The question of whether these structures,
similar to halofenate 6, also possess comparable properties with regard to the
lowering of hyperuricaemia, has not yet been elucidated.
4. wAryloxyhydratropic Acid Derivatives

The class of a-aryl0xy hydratropic acid derivatives (see Table 4) combines the
characteristics of the clofibrate and the halofenate structure. Surprisingly, this
slight modification of the basic structure caused a striking increase in activity.
Compound 47, structurally most closely related to clofibrate, showed a spectrum
of activity, which was superior to that of clofibrate (factor 5-10). Impressive
also was the higher activity of compound 53 against Su-13.437 2. The advantage
of compounds 4 8 - 5 2 was similarly clear. Several representatives selected from this
series were prepared for a clinical trial.
5. Bisaryloxyacetic Acid Derivatives

The data akeady known from the literature showing that compounds of this
structural class are 8 - 1 0 times more active than clofibrate were confkmed. The
results obtained with Sail 42-348 7, 54 and 57 showed a comparable activity of
the three compounds with slight advantage of Sail 42-348. It is also possible in this
structural field to substitute one or both chloroatoms of the Sandoz compound 7
with heterocyclic moieties to preserve the activity. Because of its high activity
119
E. Schacht
and weak acute toxicity the ethyl acetate 56, substituted unsymmetrically by
4-chlorophenol (clofibrate moiety) and 4-(4-chlorophenoxy)-phenol (HCG-004
moiety), and the corresponding free acid 55 are of particular interest. Their lipid
lowering activities were comparable to that of Sail 42-348, but some advantage was
found with 55 with regard to the influence on serumtriglyceride levels. More extensive investigations concerning side effects and long-term toxicology are planned
with these compounds.
6. Aryloxyalkanol Derivatives
It is a novel finding that in special cases the alcohols and their derivatives, obtained
by reduction of the corresponding active carboxylic acid derivatives,also possess
antihyperlipaemic activity. These qualities were found with all the compounds
listed in Table 6. Compounds 59 and 60 deserve particular mention as their
activities are by far superior to clofibrate and equal those of the corresponding
acid HCG-004 5. There exists some indication that this is an intrinsic effect of the
compounds and not the effect of the acid 5 produced by biotransformation. This
finding is sufficiently important to deserve more detailed investigation.
Since the development of many promising lipid lowering drugs was interrupted
because of lack of clinical efficacy or unfavorable side effects, it will be important
to give more and earlier attention than before to the toxicological properties which
are of special importance in drugs for a long-term therapy. Beneficial side effects,
for instance a fibrinolytic effect or a platelet aggregation inhibition, will become
more important in the future. Extensive research has been continued all over the
world, and this has increased our knowledge about both genetic and environmental
factors inducing hyperlipidaemia. It is a realistic goal for the research programs in
the next years to clarify the pathogenesis and etiology and to establish a specific
therapy for the various forms of hyperlipidaemia.
Acknowledgement. 1 wish to thank Mr. F. Krug, Mr. G. Lauterbach, Mr. G. Michelarid Mr.
W. Schumann for their skillful technical assistance.
120
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122

71) Kritchevsky, D., Tepper, S. A.: Arzneim. Forsch. 23, 6, 858 (1973)
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Received September 28, 1976
123
Prof. Dr. Prakash C h a n d r a

Gustav-Embden-Zentrum der BiologischenChemie,
Abteilung for Molekularbiologie, Theodor-Stern-Kai 7, 6000 Frankfurt 70, Germany
Dr. G e o r g e J. Wright
MerrelI-National Laboratories, Division of Richardson-MerreUInc., Cincinnati, Ohio 45215, U.S.A.
Table of Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . .
2. Chemistry of Tilorone . . . . . . . . . . . . . . . . . .
3. Toxicological Evaluation of Tilorone Hydrochloride . . . . . . . .
4. Disposition and Pharmacokinetics of Tilorone Hydrochloride . . . . .
5. Effect of Tilorone Hydrochloride on the Drug Metabolizing
Enzymes of Rat Liver . . . . . . . . . . . . . . . . . .
6. Anti.Viral Activity of Tilorone Hydrochloride . . . . . . . . . .
7. Induction of Interferon by Tilorone Hydrochloride . . . . . . . .
8. Effects of Tilorone Hydrochloride on Humoral and Cell-Mediated
Immunity . . . . . . . . . . . . . . . . . . . . . .
9. Macromolecular Interactions and Their Biological Consequences . . . .
9.1.Influence of Tilorone Hydrochloride on the Secondary
Structure of DNA . . . . . . . . . . . . . . . . . .
9.2. Mode of Tilorone Hydrochloride Interaction to DNA . . . . . .
9.3. Effect of Tilorone on the Template Activity of Nucleic Acids
9.4. Studies with Tilorone Congeners . . . . . . . . . . . . .
10. Some Future Prospects . . . . . . . . . . . . . . . . . .
11. Addendum
. . . . . . . . . . . . . . . . . . . . .
12. References
. . . . . . . . . . . . . . . . . . . . .
126
126
127
128
129
130
131
132
133
133
135
139
140
144
144
147
P. Chandra and G. J. Wright

1. I n t r o d u c t i o n
Krueger and Mayer 1) made the initial observations that tilorone hydrochloride was
a broad-spectrum, orally active antiviral agent in mice. They described activity
against several DNA and RNA viruses including Semliki Forest virus, vesicular
stomatitis virus, herpes simplex and others. Mayer and K_rueger2) described the
stimulation of synthesis of a protein in mice which fulfills the criteria necessary to
characterize it as interferon. Tilorone has subsequently been shown to influence the
immune mechanismsa), to have antitumor activity4-7), and to have anti-inflammatory properties 8).
This review is intended to summarize the information currently available in the
published literature or information which has been presented at numerous scientific
forums. Since the literature is so voluminous, the authors will not attempt to cite
every paper but will, instead, document the discussion to the extent that those who
have a deeper interest can extend the information presented in this review.
2. C h e m i s t r y of T i l o r o n e
The chemistry of tilorone hydrochloride and related compounds have been presented
in a series of five papers by Sill et al. 9), Andrews et al. 10), Albrecht et al. l t), Grisar
et al. 12), and Sill et al. 13)
The compounds found to have potent antiviral properties have two features in
common 14):
1. Two side chains containing basic (amine) functions.
2. A lipophilic central ring system of an aromatic or heteroaromatic type.
Monobasic derivatives do not yield potent antiviral agents, nor do they induce
interferon when compared to the corresponding bis basic derivatives. Removal of
the amine terminus results in loss of antiviral activity. Maximum activity is observed
with no less than 3-rings in the central aromatic or heteroaromatic nucleus.
The central ring systems found to yield antiviral compounds have included
fluorene (fluorenone), dibenzofuran, dibenzothiophene, fluoranthene, anthraquinone,
acenaphthene, xanthene, thioxanthene, phenothiazine, carbazole, phenanthrene and
others. The side chains were represented by basic ethers, basic ketones, basic esters
plus carboxamides, sulphonamides, alkanols, methylene and others attached to the
various ring systems. The amine function was usually substituted to the tertiary amine
with various alkyl substituents although a few ring types (e.g., pyrrole or piperidino)
were synthesized.
Tflorone hydrochloride, 2,7-bis [2-(diethylamine) ethoxy]-9H-fluorene-9-one
dihydrochloride, is an orange solid which is highly water soluble at neutral and acidic
pH. The compound is anhydrous and melts between 234-234.5 ~ with decomposition. The molecular weight is 483.47. Tilorone.HCl has an intense absorption
band at 270 nm in water. The pKa of the amine functions are 8.64 and 9.27,
respectively; the compound is stable in acid or base. The synthesis of tilorone
hydrochloride was outlined by Andrews et al. 1o), and Gaur and Wacker is).
126
Tilorone Hydrochloridc:The Drug Profile

3. Toxicological Evaluation o f Tilorone Hydrochloride
Preliminary toxicologic evaluation of tilorone hydrochloride has been reported by
Rohovsky, Newberne and Gibson 16) in mice, rats, dogs and monkeys. The 24 hr
oral LDs0 was 959 mg/kg for mice and 852 mg/kg for rats. In subchronic studies,
oral doses up to 180 mg/kg/day in rats and mice produced a dose-related depression
of body weight gain and food consumption. In dogs and monkeys oral administration
of _> 20 mg/kg/day produced clinical signs of anorexia, emesis, ptosis, salivation,
ataxia and tremors. Oral doses of 3, 10 or 30 mg/kg/day in pregnant rabbits during
days 9 through 16 of gestation produced no teratologic effects.
Rohovsky, Newberne and Gibson 17) reported the appearance of vacuolation
and granulation of peripheral leucocytes in the mouse, rat, dog and monkey. A
concomitant alteration in the reticuloendothelial system also occurred which
consisted of intracytoplasmic accumulation of ovoid structures with vacuolated
centers in Kupffer cells of the liver and mixed macrophages of the spleen and lymph
nodes. Smears of bone marrow revealed abnormal granules in myeloid cells as
primitive as neutrophilic myelocytes, but the myeloid:erythoid ratio was not
altered. The post-treatment regression of these effects was species dependent.
Zbinden and Emch is) extended the observations of Rohovsky et al. 17), on the
effect of tilorone hydrochloride on the peripheral blood of laboratory animals. They
described in rats, a marked, but transient, lymphopenia after single oral doses of
100 mg/kg. Recovery of the lymphopenia started after 12 hr and was accompanied
by depletion of small lymphocytes in lymph follicles of the spleen and the appearance
of y.oung lymphocytes with vacuoles and basophilic granules in smears of lymph
nodes and spleen. Abnormal mononuclear cells whose protoplasm often contained
vacuoles and basophil granules appeared in the blood after 48 hr and disappeared
in the course of 2-3 weeks. Repeated dosing caused only minor hematologic
changes and affected no other organs.
Levine, Gibson and Mege119) studied the depletion of the lymphocyte population
caused by tilorone hydrochloride given orally or subcutaneously to rats and mice in
spleen, lymph nodes and Peyer's patches. Although the thymus itself was not affected,
thymus-dependent areas were affected similarly to that caused by neonatal thymectomy,
thymic aplasia, or antilymphocytic serum. The effects of tilorone were not
dependent on the presence of adrenal, thymus or spleen, and the depleted tissues
were rapidly repopulated. The previously sensitive areas were resistant to further
doses of the same drug.
The effect of tilorone hydrochloride on the ability of platelets to aggregate was
studied by MacKenzie and Schatzman 2~ In vitro, tilorone hydrochloride did not
inhibit the first phase of human platelet aggregation induced by ADP but was a
potent inhibitor of the second phase of platelet aggregation (release reaction)
caused by epinephrine. In rats given two 100 mg/kg 24 hr apart, collagen-induced
aggregation was inhibited in platelets obtained 24 hr after the last dose. These
results are consistent with inhibition of the second phase platelet aggregation
(release reaction) similar to that seen with other non-steroidal anti-inflammatory
compounds. With the exception of aspirin, the effect from tilorone is of longer
duration.
127
P. Chandta and G. J. Wright

4. Disposition and P h a r m a c o k i n e t i c s o f Tilorone H y d r o c h l o r i d e
Wacker et al. 21), reported the distribution of 14C-tilorone in mice 16 hr after
intraperitoneal injection of 1.5 mg in phosphate buffered saline. Higher concentrations of radioactivity were found in liver followed by spleen, kidney and lung.
Relatively lower concentrations were found in thymus, heart, adipose tissue, skeletal
muscle and blood. A large proportion of the radioactivity isolated from liver
homogenates and urine was unchanged tilorone. Two unidentified products were
seen in addition to tilorone. A significant amount of the radioactivity was associated
with the sediment following centrifugation. Since Triton X-100 was needed to
release some of the radioactivity, binding to membrane particles was suggested.
Nucleic acids extracted by phenol contained no radioactivity.
The metabolic disposition of 14C-tilorone hydrochloride in mice, rats and dogs
has been studied by Hook et al. 22). In contrast to the studies reported by Wacker
eta/., 2D, all doses were administered orally. In the Long-Evans rat, elimination of
a radioactive dose (100 mg/kg) of tilorone hydrochloride was about evenly divided
between urine and feces. Elimination was biphasic with the tll: of the first phase
1.3 days and the h/2 of the second phase 4.2 days.
Six days after dosing, 40-45% of the dose remained in the rats. Tissue radioactivity was located principally in the liver (8%), lung (1-2%), spleen (1-2%),
gastrointestinal tract and contents (4-5%) and the carcass (20-24%). Tissues having
the highest concentrations of radioactivity six days post-dose were spleen, liver,
lung, eyes, adrenals and lymph nodes. Concentrations in heart, skeletal muscle and
brain were relatively low, while blood and plasma concentrations were very low.
Concentrations of radioactivity in the liver reached a maximum at 3 hr post-dose,
in the lung at 4 hr and was still increasing in the spleen at 6 hr. These results imply
rapid absorption with subsequent redistribution.
Elimination of radioactivity and of material with similar UV absorption pro.
perties to tilorone occurred with a half life of 3.5 :-4 days in.mice after a dose of
250 mg/kg.
In beagle dogs, urinary excretion accounted for 17-21% of the dose (20 mg/kg)
during 7 days following dosing. Excretion by way of feces accounted for 13-14%
of the dose. Only 8-9% of the dose appeared in feces during the first 3 days, which
suggested that absorption was at least 90%, if not 100%, complete. Seven days postdose, radioactivity was located primarily in the liver (22-24%), small intestine and
contents (5-6%), lung (2.7-3.0~) and spleen (1.5-1.7%). Highest concentrations
of radioactivity were present in liver, spleen and adrenals. Lower levels were found
in heart, brain and skeletal muscle. Low plasma concentrations of radioactivity,
in comparison with tissue concentrations, resulted in extremely high tissue-plasma
ratios. Ratios exceeding 1000-to-1 were found for liver, spleen, adrenals and pancreas.
High concentrations of radioactivity occurred in the pigmented structures of the
eye, the choroid and iris, and appreciable concentrations in the retina. Concentrations in all other eye substructures were quite low suggesting that binding to
melanin containing substructures had occurred.
Tissue metabolites from the rat were examined and identified. Thin-layer
chromatography and autoradiography revealed the presence of 6 - 8 radioactive
128
Tilorone Hydrochloride:The DrugProfile

compounds. Unchanged tilorone represented the major portion of the radioactivity in rat tissues, even at 6 days. Mono-de-ethylated tilorone was found in the
highest quantities of any of the metabolites and represents the major metabolite.
Di-de-ethylated tilorone with one ethyl group removed from each side chain was
found in considerably lower amounts than the mono-de-ethylated product. Reduction
of the carbonyl resulted in the fluorenol analog of tilorone. Polar metabolites were
found to be the mono-N-oxide and di-N-oxide analogs of tilorone. Metabolism of
tilorone proceeds by N-dealkylation, N-oxide formation and carbonyl group
reduction.
Gaur and Chandra2a) extended their earlier studies in mice with 14C-tilorone.
Following an intraperitoneat injection of 20 mg/kg in male AKR-mice, the halflife of elimination was about 72 hr with the major excretory route by way of the
kidney. In confirmation of earlier findings, spleen and kidney had the highest
specific activity. Distribution of radioactivity 24 hr after the dose was: liver, 25%;
spleen, 2.5%; kidney, 2.3%; lungs and pancreas, about 1.5% each; and less than
0.5% of the administered dose in each of the remaining tissues.
Subcellular distribution of tilorone hydrochloride has been examined. Gaur and
Chandra 24) studied the subcellular distribution of radioactivity in liver, spleen, brain,
lungs and kidney of mice and liver, spleen, brain, kidney and heart of rats after a
dose of 50 mg/kg of 14C-tiiorone by intraperitoneal injection. A substantial proportion
of the radioactivity was found in the fraction which sediments at 700 x g and
contains, in addition to cell debris, the nuclei. The remaining radioaciivity was
distributed between the mitochondrial, microsomal and supernatant fractions. The
authors suggested that the primary target of tilorone hydrochloride may indeed be
the molecular species localized in the nuclei; Le., the DNA.
The studies of Gaur and Chandra24) were extended by Leeson et al. 2s). Since
Chandra eta/. 26-2s), had described the in vitro binding of tilorone to DNA and
later extended these observations, the studies of Leeson et al. 2s), were specifically
designed to study the localization of tilorone and one of the metabolites in the
nucleus of rat liver. Equilibrium dialysis studies showed that binding occurs with
both plasma and tissue homogenates with stronger binding by the tissues. In
confirmation of the findings reported by Gaur and Chandra 24), the 700 x g ppt.
of rat liver homogenate contained the major fraction of drug related material.
However, intact nuclei isolated from the liver homogenate contained proportionately
less drug related material even though the DNA was relatively concentrated. These
in vivo findings suggest that tilorone does not selectively localize in the nuclei. Instead,
the concentration of tilorone in the various subcellular fractions appeared to relate
better to protein concentration suggesting a relatively non-specific binding throughout
the cell.
5. Effect o f Tilorone Hydrochloride on the Drug Metabolizing

Enzymes o f Rat Liver
In companion papers, Renton and Mannering29) and Leeson et al.3~ described the
decrease in activity of the hepatic cytochrome P450 mono-oxygenase system following
129

oral administration of tilorone hydrochloride (doses ranged from 20-250 mg/kg.)
Hexobarbital sleeping times were prolonged and blood levels of hexobarbital were
elevated after doses of tilorone. Zoxazolamine paralysis times were prolonged after
four doses of I00 mg/kg/day but not after a single dose. Concentrations of eytochrome
P450 and NADPH-cytochrome c-reductase were reduced as were various enzyme
activities related to the mono-oxygenase systems. Microsomal protein concentrations
were initially reduced but had recovered to control levels with 21 days of continuing
treatment with 100 mg/kg/day. Aminopyrine demethylase and hexobarbital oxidase
remained decreased with 21 days of continuing treatment. Similarly, cytochrome
P450 concentrations remained decreased. Electron micrographs of rat liver, after
treatment with tilorone hydroehloride 100 mg/kg/day for 21 days, revealed many
membranous structures in the form of whorls in the cytoplasm of the cell. It was
postulated that the whorls originated from the endoplasmic reticulum in an attempt
by the liver to restore full enzymatic capabilities in the face of continued drug
administration.
Tilorone hydroehloride was not a direct inhibitor of the mono-oxygenase system
of rat liver. In vitro addition of tilorone hydrochloride did not affect microsomal
drug metabolism nor did it affect cytochrome P450 contents of the microsomes.
The rate of incorporation of S-Amino (3H) levulinic acid into cytochrome P450
was not affected by tilorone 9HCI.
6.
Anti-Viral A c t i v i t y o f Tilorone H y d r o e h l o r i d e
Krueger and Mayer 0 first described tilorone as a broad spectrum, orally active
antiviral agent. In the first publication, activity against Semliki Forest, vesicular
stomatitis, encephalomyocarditis, Mengo, vaccinia, herpes simplex and three strains
of influenza viruses in mice was described. These authors 2) attributed the antiviral
activity of tilorone largely to the induction of interferon in the treated mice. Mayer
and Krueger3~) later described antiviral activity against Semliki Forest virus in rats
and eye lesions caused by herpes simplex in rabbits. Activity of tilorone against
herpesvirus hominis, type 1 and 2, in a rat and mouse tail lesion test was described
by Yoshimura et al. 32).
In subsequent reports, tilorone hydrochloride prolonged the survival time of
mice infected with Friend leukemia v i r u s 3 3 ) , protected Swiss mice against 6 intracerebral or 6 subcutaneous LDso'S of the CVS strain of rabies virus infected 19 h after
treatment with drug34) , and prolonged the survival of mice infected subcutaneously
by the "slow virus" scrapie as). Hofmann and Kunz 36) described the protective effect
of tilorone hydrochloride on experimental tick-borne encephalitis in mice. Mice
infected with 80 LDs0 foot-and-mouth disease virus, type 0, were protected by
250 mg/kg of tilorone given orally 37). Tilorone hydrochloride prevented the production of serum antibody to live Venezuelian equine encephalomyelitis vaccine in mice
presumably because of inhibition of viral replication by interferon production 3s).
Mayer, Bray and Camyreag) suggested, however, that not all of the activity against
viruses can be attributed to the induction of interferon production. Direct virus
130

inactivation was demonstrated and topical activity against herpes-induced lesions
on mouse skin or in the rabbit eye was found.
7.
Induction of Interferon by Tilorone Hydrochloride
Mayer and Krueger 1) first described the appearance of an antiviral serum component
in mice treated orally with tilorone hydrochloride which full'flied sufficient biological
criteria to be classified as an interferon. Krueger et al. 4~ described the interferon
induction properties of a number of analogs of tilorone and described the hyporesponsiveness which occurs with tilorone as well as other antiviral agents. The
interferon produced by treatment of mice and rats has been characterized by Camyre
et al. 4D, and Camyre and Groelke42). Serum from tilorone or poly I: C treated
mice was found to possess an interferon with a single molecular species with a
molecular weight by Sephadex G-100 chromatography of 34,000. Serum interferon
from tilorone-treated rats was associated with two distinct molecular species of
27,000 and 80,000. The interferons were resistant to ribonuclease and to heat. Some
differences between rat and mouse interferons were described; e.g., sensitivity to
pH 2.5.
Administration of cycloheximide (60 mg/kg) 1 hr prior to tilorone administration
inhibited the interferon response 43'44) . The inhibition by cycloheximide suggested
that protein synthesis was involved in the appearance of interferon in the serum. For
a more complete discussion of interferon induction, see the review on synthetic
interferon inducers written by DeClercq4s).
In discussing the mechanism of antiviral protection and stimulation of interferon
production in the mouse, DeClercq and Merigan 46) concluded that there was a direct
relationship between the extent of protection against vesicular stomatitis virus, the
titers of interferon produced and the doses of tilorone. Giron et aL 47), however,
found no correlation between interferon induction and protection against MM virus
in mice. Protection was achieved at doses far below the doses at which detectable
interferon was found in the serum. Both findings may be consistent with differing
mechanisms of viral inactivation for the two viruses under study.
Although interferon could be readily induced in mice and rats by tilorone,
attempts to induce interferon in monkey and humans have not been successful4s'49).
Interferon induction in normal and leukemic lymphocyte cultures with tilorone
has been observed s~ The interferon response observed in normal lymphocyte
cultures appeared to be correlated with the toxic effect of tilorone. The effect
observed in leukemic cultures required higher concentrations of tilorone, but,
similarly, appeared to be related to cell viability. Tilorone has been reported to
stimulate production of interferon by itself in mouse embryo fibroblasts and, in
combination with poly rl :poly rC/DEAE.dextran in mouse L929 cellssl) . Human
foreskin fibroblasts were not stimulated. The degree of synergism between tilorone
and the nucleotide-dextran complex was proportional to the concentrations of
tilorone and poly rI/poly rC and was influenced by the times of addition of the
compounds relative to each other.
131

8. E f f e c t s o f Tilorone H y d r o c h l o r i d e o n H u m o r a l and Cell-Mediated
Immunity
Hoffman et al. s2), presented evidence that a single oral dose of tilorone enhanced
the primary immune response to sheep red blood cells (SRBC) in mice as measured
by the Jerne Plaque technique. They also reported an increase in hemolysin titer
after tilorone administration. To further evaluate the action of tilorone on humoral
antibody responses, Megel e t al. 3) have studied its effect on 19S and 7S production
in the primary and secondary immune responses in mice. It was 'found that
tilorone elevated 19S antibody titer on days 3 and 4 after immunization. After 9 days
of continuous drug administration, the 19 S response for both groups was diminished
compared to days 3 and 4; however tilorone was found to cause a significant increase
in the 7S antibody production compared to controls. Tilorone also stimulated the
19S response to E. coli endotoxin, a thymus-independent antigen, on days 3 and
4 after immunization.
The effect of tilorone on 19S and 7S antibodies was also measured in the
secondary immune response 3). In this study the mice were immunized with SRBC
at day zero. Tilorone was administered at a dose of 50 mg/kg subcutaneously starting
on day 20. A second immun~ation was given at day 21 and tilorone was given daily
until day 23 (3 days after challenging). Both 19S and 7S responses were significantly
increased compared to control.
Besides IgG (7 S) and IgM (19 S) production, tilorone was also found to elevate
IgE levels. Using a parallel line assay, as described by Finney 53), Megel e t a t 3) found
that tilorone elevated IgE-like antibody levels 3.2 times with relation to saline control.
The data show that the drug serves as an adjuvant for a variety of immunoglobin
classes (IgG, IgM and IgE antibody production). The effects on IgA responses to
antigenic stimulation remain still to be determined.
In contrast to the effects of tilorone on humoral antibody production, the drug
suppressed cell-mediated immune responses as evidenced by the significant decrease
in paralysis in the EAE (experimental allergic encephalomyelitis) model, the
inhibition of the tuberculin skin reaction, and the reduction in the secondary
swelling in adjuvant arthritis 3).
Tilorone appears to be very selective in its action in that, it enhances humoral
antibody production while suppressing delayed hypersensitivity response. It differs
from the well-known immuno-suppressive compounds (e.g. glucorcorticoids, antimetabolites etc.), which are capable of suppressing both. More recently, other
synthetic compounds having selective effects on the immune systems have been
reported. Freedman et al. 54) and Fox e t al. ss) have reported that oxisuran suppresses
skin-graft rejections in mice, rats and dogs but has no effect on antibody production.
Renoux and Renoux s6) have shown that levamisole enhances cell-mediated immunity
as evidenced by its effect on graft vs. host reactions. Thus, the results with tilorone
and some other drugs suggest that, the immune system can be modulated selectively
by pharmacologic manipulating.
132

9.
M a c r o m o l e c u l a r I n t e r a c t i o n s a n d T h e i r Biological C o n s e q u e n c e s
9.1. Influence of Tilorone Hydrochloride on the Secondary Structure of DNA
The possibility that this compound may react directly with DNA was indicated by
the cytogenetic studies of Green and West sT). Tilorone was found to inhibit mitosis
significantly at 3.0/~g/ml, and produced chromosomal abnormalities at 1.5/ag/ml.
Soon it was discovered by Chandra e t a/.26-28)that tilorone does form molecular
complexes with DNA and polydeoxynucleotides. Some of these studies will be
described here.
Interaction between nucleic acids and biologically active compounds may induce
changes in the electronic spectra of the components. Tilorone hydrochloride in water
shows two absorption maxima, in the ultraviolet region around 271 nm, and in the
visible region around 470 nm. Thus the investigation of the long wavelength band,
where DNA and RNA do not absorb, should provide some evidence whether or not
the chromophore of tilorone hydrochloride is involved in the binding process.
0.15
0.10
g
,.Q
<
0.05
....
350
400
450
Wavelength (nm)
500
550
Fig. 1. Effect of calf thymus DNA on the visible absorption spectrum of tilorone hydrochloride.
Samples contained 4.25 x 10-4 M of tilorone hydrochloride, 0.01 M Tris-HC1(ph 7.0) and
DNA at 0.5 x 10-3 M (+
+); 1 x 10-3 M (o - - o); 2 x 10-3M (*
*). No DNA was
added to the sample ( A - - A )
133

Figure 1 depicts the absorption spectrum ( 3 5 0 - 5 0 0 nm) of tilorone hydrochloride alone (continuous line with triangles) or in the presence of various
amounts of calf thymus DNA. There is a characteristic change in tilorone spectrum
in the presence of DNA. In the presence of calf thymus DNA the visible absorption
spectrum of tilorone hydrochloride is depressed and red shifted. This hypochromic
effect of DNA on the absorption of tilorone chromophore is dependent on DNA
concentration. The largest hypochromic effect is observed at 2 x lO-3M DNA-P
in a 4.25 x 10-4M solution of tilorone hydrochloride.
The concentration-dependent effect of calf thymus DNA on the visible absorption
spectrum of tilorone hydrochloride indicates that the tilorone chromophore interacts
with DNA. Figure 2 depicts the visible absorption spectra of tilorone alone (curve
one), or in the presence of yeast RNA (curve two), denatured DNA (curve three)
and native double-stranded DNA (curve four). The visible spectra indicate that at
equimolar concentrations, DNA in its double helical state produced largest changes
0.11
0.10
0.09
0.08
0.07
0.06
/, 0.05
<
0.04
0.03
0.02
0.01
0
350
400
450
500
Wavelength (nm)
550
Fig. 2. Effect of native calf thymus DNA, denatured calf thymus DNA and yeast RNA on the
visible absorption spectrum of tilorone in 0.01 M Tris-HC1(pH 7.0). Curve 1 is the spectrum of
free tilorone (4.25 x 10-4 M). Other curves depict the spectra of tilorone in the presence of
yeast RNA (curve 2), denatured DNA (curve 3) and native DNA (curve 4). Molar concentrations of nucleic acids (2 x 10-3 M) refer to phosphorous content of the polymer
134

in the absorption spectrum of tilorone, whereas the effect of single.stranded DNA
is slightly weaker. In contrast, the yeast RNA exerts only a slight effect on the
visible spectrum of tilorone hydrochloride. These data indicate a specificity of the
tilorone chromophore towards DNA.
Further information on the binding of tilorone with DNA was derived by studying
the thermal melting of the complex 27'2s). In order to characterize the stability of
DNA secondary structure in the presence of tilorone, temperature profiles were
run at tilorone/DNA-P molar ratio of 1 : 5. Tilorone hydrochloride shows a large
increase in the thermal transition temperature (Tm) of native DNA; the Tm of calf
thymus DNA was raised from 71.6 to 85.2 ~ under these conditions.
9.2. Mode o f Tilorone Hydrochloride Interaction to D N A
Hypochromic effect of native DNA on the absorption of tilorone chromophore is

partially reversible by Mg 2+ ions. Figure 3 depicts the absorption spectra ( 3 5 0 550 nm) of tflorone hydrochloride alone, 4.25 x 10 - 4 (curve one), in the presence
of 4 x 10- 3 M DNA-P (curve four) containing 0.01 MgCI2 (curve two) or 0.1 M
NaC1 (curve three). It follows from these results that the DNA-drug interaction is
0.10
0.09
0.08
0.07
0.06
o 0.05
.<
0.04
0.03
0.02
0.01
0
350
400
450
500
550
Wavelength (mlt)
Fig. 3. Effect of Na+ and Mg 2+ on the visible absorption spectrum of the tilorone-DNA
complex. Samples contained 4.25 x I0-4M tilorone, 4 x 10-3M DNA-P, 0.01 M Tris-HCl
(pH 7.0) and 0.01 M MgCI2 (curve 2) or 0.1 M NaC! (curve 3). Curve 1 is the spectum of
.
.
.
free tilorone;
curve 4 .is the spectrum
of the tilorone-DNA complex m the absence of Na+ and Mg2+
135

very sensitive to magnesium ions. The effect of magnesium ions on tilorone binding to
DNA was confirmed by density-gradient studies using labeled tilorone hydrochloride28).
These studies indicate that electrostatic forces contribute greatly to the binding
process. The interaction between tilorone and DNA may, however, involve other
kinds of forces. Tilorone forms a reversible complex with DNA, since the drug
could be completely dissociated from a DNA-cellulose column. Interaction of
apurinic and apyrimidinic DNA's with tilorone hydrochloride also gave spectral
changes. However, only with the apyrimidinic DNA, the spectrum of the bound
drug was similar to that found with native DNA.
The absorption spectrum studies presented above merely reflect the electronic
environment of the molecule and do not give specific information about the type
of interaction. The data which must be accounted for in considering a physical mode
for the binding process can be derived from several different approaches. Hydrodynamic measurements on the DNA-drug complex are of interest, since Lerman s8, sg)
has established that an increase in the intrinsic viscosity of DNA and a decrease in
the sedimentation coefficient of the polymer are two criteria for intercalation of
ring systems between base pairs of a double-helical DNA.
The relationship between the intrinsic viscosity of DNA and the amount ("r")
of bound tilorone was studied 28). The intrinsic viscosity of the complex increases
with r up to a limiting value of about 0.05. The maximum relative enhancement of
viscosity was about 1.7. In addition, at the same ionic strength and at a ligand to
DNA-P molar ratio of 0.1, the sedimentation rate of DNA was decreased to 78% of
the value in the absence of ligand.
These observations are consistent with an intercalative mode of binding in the
interaction of tilorone hydrochloride with double-helical DNA. These results were
not examined in an attempt to verify whether they agree with measurements of the
length increase on sonicated DNA. For this reason, the intercalation model of the
DNA complex remains tentative.
The interaction of tilorone hydrochloride with native DNA stabilizes the double
helical structure of the macromolecule towards thermal denaturation. The effect
of tilorone hydrochloride on the thermal denaturation of DNA's from various
sources having different base composition has been studied 2s). At a drug to DNA-P
molar ratio of 0.21, the ATm increased with increasing AT content of the DNA. This
observation indicates that tilorone hydrochloride perferentially binds to the dAT
portions of the DNA molecule. This is confirmed by the strong effect of tilorone
hydrochloride on the thermal transition temperature of poly d (A-T), ATm = 29 ~
An intercalative mechanism for binding of a ligand to DNA is consistent with
a stabilization of the double helix. Such a stabilization, however, does not constitute
proof of intercalation. But, when considered with the evidence of the results reported
here, showing increased viscosities and decreased sedimentation rate of DNA, one
may conclude that the large increase of Tm points to an intercalative mode of
binding.
The AT-specificity in the binding of tilorone to DNA was also observed in the
quantitative equilibrium binding measurements. The equilibrium binding data were
plotted as r/(u) vs. r, where r is the moles of bound tilorone divided by the DNA
concentration in base pairs 6~ and (u) is the concentration of unbound tilorone.
136

From this plot binding parameters were obtained appropriate to a model in which
all DNA binding sites are considered to be independent of each other 61). The
equation from this model is:
r/(u)=Kapp(Bapp-r),
where Kapp and Bapp are the apparent binding constant and number of binding sites
per base pair, respectively; Kapp is the negative of the slope, and Bapp the r/(u) = 0
intercept of the linear region of the r[(u) vs. r plot.
The Scatchard plots for the binding of tilorone to calf thymus DNA (Fig. 4a),
Mic. lys. DNA (Fig. 4b), poly (dA-dT).poly (dA-dT) (Fig. 4c), and poly (dG-dC) 9
poly (dG-dC) (Fig. 4d) are shown in Fig. 4. As follows from the Scatchard plots
for natural DNAs, (Fig. 4a and 4b), the independent site model does not fit for
DNA-ligand interaction; however, we employed the binding parameters of this
model to analyse the Scatchard plots. The binding parameters, gap p and Bapp
derived from the Scatchard plots (Fig. 4a-d) are presented in Table I. This table
summarizes the results of several measurements.
As seen in Table 1 the apparent binding constants for both the natural DNAs
are of the same magnitude, Whereas the Kapp for poly (dA-dT) 9poly (dA-dT) is
higher by a factor of two. The Kapp value for poly (dG-dC) 9poly (dG-dC) is less
by a factor of 10 - 2 , compared with those of natural DNAs and the synthetic
polydeoxynucleotide poly (dA-dT) 9poly (dA-dT). The data on the number of
binding sites per base pair (Bapp) for various DNAs show a strict correlation with
the AT-content of the biopolymer. On the basis of the Bapp data, the closest
distance between bound tilorone molecules is four base pairs. The values shown in
Table 1 give almost a linear curve, showing a linear dependency of Bapp on the AT
Table 1. Binding constants for the interaction of tilorone hydrochloride with DNAs and
synthetic polydeoxynucleotides
Source of DNA
A.
Natural DNAs
Calf thymus
Mie. lysodeikticus
B.
Synthetic polymers
Poly (dA-dT)-poly (dA-dT)
Poly (dG-dC)-poly (dG-dC)
Bapp
Kapp
M-1
0.16
0.12
0.066
0.062
2.9
4.1
5.5
6.0
0.25
0.25
1.02 x 106
1.04 x 106
6.9 x 103
x l0 s
x 105
x 105
10 s
All experiments were carried out at 200 in 0.1 M Tris-HCl (Ph 7.0). The binding parameters
were derived from the Scatchard plots (Fig. 4, a-d); the experimentswere carried out by
equilibrium dialysis as described under Fig. 4. Kapp = apparent binding constant; Bapp =
number of binding sites per base pair.
137
\
4 - -~~
(a)
3 -~4~
(b)
b
-6
E
3-
.--,
oE
2-
"IZ
e+
4-0
O0
0.05 0.10 0.15 0.20

r
0.05 0.10
r
0.15
30
20
"6
E
0E
,I-x
I
00
(d)
+ 9
0j~
01~
0.05 0.10 0.15 0.20

r
2- 9
..~o e _
+ 0
--+~-------o~
0
4-
qV_
0(~
0.05 0.I0 0.15 0.20 0.25

r
Fig. 4. Scatchard plots for the binding of tilorone hydrochloride to calf thymus DNA (a);Mic.
lysodeiktieus DNA (b); poly (dA-dT) 9poly (dA-dT) (c); and poly (dG-dC) 9 poly (dG-dC) (d).
Each different symbol corresponds to a separate experiment. Thus, each figure represents a set
of 4 or 5 separate experiments, t is moles of bound tilorone/base pair concentration and (u) is
the concentration of unbound tilorone. Equilibrium dialysis was carried out by a procedure and
an apparatus (Dianorm, supplied by Dr. Virus KG, Bonn, Germany) described by Weder et al. 67).
Dialysing membrane (0.025 mm thick) was sandwiched between t~,o halves of a Teflon
(round) macro-ceU (dialysable volume = 1 ml). The DNA, or labelled tilorone solutions were
introduced by separate micro syringes on either side of the membrane through the side valves.
The valves were closed air tight and the macro-ceUs were f'LXedinto a rotating machine. All
equilibrium dialysis studies were carried out at 20*, and at 10 rotations/rain. Under these
conditions equilibrium was attained in 4 - 5 hr. After the equilibrium was reached 0.8 ml of
the solution from either side of the membrane was withdrawn by microsyringes and the
radioactivity was determined using dioxan scintillation fluid
138

content of DNAs. The value for poly (dG-dC). poly (dG-dC), was extrapolated to
zero, since the Scatchard plot (cf. Fig. 4d) does not indicate any specific binding
of tilorone to this polymer.
9.3. Effect of Tilorone on the Template Activity of Nucleic Acids

The interaction of tilorone hydrochloride to DNA encouraged us to study the
template activity of the complexes in DNA- and RNA- polymerase systems from
E. coil Both the activities were found to be inhibited by tilorone; the DNA polymerase reaction being more sensitive towards tilorone. The inhibition of the RNApolymerase reaction by tilorone was dependent on the A-T content of DNA-template.
The template activity of poly (dA-dT) in the RNA polymerase reaction is distinctly
more sensitive towards tilorone than that of DNA; particularyly, at low drug concentrations the poly (dA-dT)-catalyzed reaction was three times more sensitive than the
DNA.catalyzed activity of RNA polymerase reaction 6~
The nature of the inhibitory response, as depicted in Fig. 5, indicates that these
compounds compete for the binding sites on DNA. This is clearly shown in the
20
15
~l>
10
50
I
150
I
250
._L_
$1 [A260 ]
Fig. 5. Lineweaver-Burkplot of the effect of increasingconcentrations of template DNA on the

incorporation of 3H-AMPinto RNA by RNA polymerase ofE. coli K-12, in the absence of
tilorone (o
e), or in the presence of tilorone, 3.2 x 10-SM ( s - - o ) , Abscissa: 1/S =
(DNA temp.lalte,A26Oper reaction mixture)-1. Ordinate: I/V = (3H-labelledAMP incorporated
into RNA)139

Lineweaver-Burk plot of the kinetic data obtained by measuring the RNA polymerase activity at various concentrations of template (Fig. 5). Figure 5 depicts the
kinetic curves of the reactions in the absence of the inhibitor (o - +), and in
the presence of 3.2 x 10- s M tilorone (o
- o). This shows that tilorone is a
competitive inhibitor of DNA template activity in the RNA polymerase reaction.
9.4. Studies with Tilorone Congeners

The interaction of tilorone with DNA and synthetic polydeoxynucleotides can be
influenced by modifying tilorone structure. Such studies are indeed, important to
elucidate the role of structural entities in their complex formation with DNA.
Structure-activity relationship of tilorone derivatives (Fig. 6) has recently been
studied 7,60,62).
The effect of tilorone and congeners (Fig. 6) on the DNA-dependent RNA
polymerase reaction is shown in Fig. 7). The template activity of native DNA is
strongly inhibited by DEAP-fluoranthene, showing an 80% inhibition at a
concentration 8 x 10-6 M. Other derivatives, at this concentration do not show
any significant inhibition of the template activity of DNA. However, at higher
concentrations one observes a dose-dependent inhibition of DNA-template activity
by DEAE-fluorenone, DMAA-dibenzothiophene, DEAA-fluorene and DMAAdibenzofuran. The monosubstituted derivative, MEAA-fluorene does not show
any activity, even at higher concentrations.
The inhibition of DNA template activity by tflorone and its congeners is strongly
influenced by substitutions in the ring (e.g. thiophene, furan, etc.), as well as in the
side chains. It was, therefore, of interest to study whether such substitutions influence
their interaction to DNA. Figure 8 depicts the melting curves of calf thymus DNA
alone (curve one), or in the presence of MEAA-fluorene (curve two), DEAA-fluorene
(curve three), DMAA-dibenzothiophene (curve four), DMAA-dibenzofuran (curve
five) and DEAE-fluorenone (curve six). These studies were carried out at a drug/DNAP ratio (r) of 0.1. Tilorone hydrochloride (DEAE-fluorenone) shows a large increase in
the thermal transition temperature (Tin) of native DNA (curve six). The congener
DEAP-fluoranthene, under these conditions, showed a very similar response (curve
not shown). It is interesting to note that DMAA-dibenzofuran, though less active
than DMAA-dibenzothiophene and DEAA-fluorene in the RNA polymerase reaction,
has a higher effect on the Tm of calf thymus DNA, than exhibited by these two
derivatives.
The structure-activity relationship observed in the RNA polymerase reaction, is
not strictly exhibited by the melting curves of DNA and congener complexes. The
latter studies were done in the absence of magnesium ions whereas, the RNA
polymerase reaction requires magnesium ions for its enzymatic activity. The fact that
compounds of tilorone type form complexes with Mg2+ may explain differences in
the structure-activity relationships of congeners in the RNA polymerase reaction,
and their effects on the melting behavior of DNA. Thus, the dibenzofuran congener,
though less active than DEAA-fluorene in the RNA polymerase reaction, has a higher
affinity for DNA than DEAA-fluorene. The dibenzofuran derivative should have a
140
O--CH2CH2--N(C2H5) 2
DEAE - Fluorenone
Ti{orone
-2HCI
O--CH2CH2--N(C2H5)2
O
II
C-- O-- CH2CH2CH2-- N(C2H5)2
DEAP - Fluoranthene
RM[-9563 DA
92 HCI
C--O--CH2CH2CH2-- N(C2H5)2
II
O
O
II
C--CH2--N(CH3) 2
2 HCI
DMAA- Dibenzothiophen
RMI - 11877 DA
C-CH2--N(CH3) 2
0
II
C-- CH2--N(CH3)2
O~
92
HCt
DMAA- Dibenzofuran
RMI- 11567 DA
C--CH2--N(CH3) 2
II
O
c/O
~--/CH2--N(C2H5)2
OEAA- Fluorene
RMI- 11002
-2HC(
C--CH2--N(C2H5) 2
II
0
MEAA- Fluorene
RM[- 1182g A
monosubstituted RM] 11002
Fig. 6. Chemical structures of tilorone and congeners
141
P, Chandra and G. J. Wright
100
<
7
O~
' ~ o r e n e
80
4O
:~
2()
<
J
\
~
"~_~"~-" ~
"
'
6
'
'
16
~
: - ~
~t
32
OEAp-Flu~
64
qe
1
96
Concentration (ItM)
Fig. 7. Inhibition of DNA-dependent RNA polymerase reaction (E. coil K-12) by tilorone and
congeners
////
. ~,,,-- ..~.~.~L~.I-~'
100
ao
oo
//.,///>
~o
//,5"-'.,
"/y
'" &
7o
4~
Temperature
a'o
&
' '
(~
Fig.8. Effect of tilorone and congeners on the thermal transition temperature (Tin) of calf thymus
DNA. Solvent is 0.01 M Tris-HCl pH 7.0 and the concentrations of DNA-P and congeners axe
5 x 10-6M, respectively. Curve 1 = DNA; 2 = DNA + MEAA-fluorene; 3 = DNA + DEAAfluorene; 4 = DNA + DMAA-dibenzothiophene; 5 = DNA + DMAA-dibenzofuran and 6 -- DNA
+ DEAE-fluorenone
higher tendency for chelating with magnesium ions, which leads to its partial
inactivation in the RNA polymerase reaction.
To measure the effect of various tilorone congeners on the oncogenic activity of
MSV (M), viral suspensions were incubated with 5 x 10-s moles/ml of each compound
at 37 ~ for I hour. In the control group, where no compound was used, virus was
preincubated with the soNent. Tris.buffer, 0.01 M, pH 7.4, 0.2 ml of this mixture
containing 1 x 1 0 - 7 moles of the drug, was injected intraperitoneally. The amount
of compound introduced this way had no direct physiological effect on the host
(unpublished results). The mortality and the survival period were signkficanfly
influenced by tilorone and two of its congeners, DEAP-fluoranthene showed a
142

significant inhibition of splenomegaly induced by FLV. DEAA-fluorene showed
only slight activity. It is interesting to note that MEAA-fluorene did not show any
activity in this system 7 ,62)
Since none of the compounds at the concentrations used showed a complete
supression of splenomegaly, one would expect a residual viral activity in spleen
extracts of mice, which received FLV suspensions preincubated with these compounds.
Studies are now in progress to evaluate the leukemogenic activity of cell-free spleen
extracts, prepared from mice inoculated with pretreated suspensions. Wu et al.6 3),
have carried out such studies with RLV and rifamycin derivatives. They reported
that the inoculation of mice with inocula from mice infected with RLV pretreated
with AF-ABDP and AF/DNF 1 did not cause splenomegaly.
If the supression of biological activity of RNA tumor viruses is due to a block
of some molecular event (s) involved in oncogenesis, one would expect an
inhibition of DNA polymerases by these compounds. That tilorone does inhibit the
DNA polymerase activity of oncomaviruses has been shown earlier 26). An attempt
was made to correlate the biological response of various congeners with their
inhibitory activity in the DNA-polymerase system of oncornaviruses. These studies
were done using purified FLV, since in our hands this system showed a good
endogenous activity.
z~
100
MEAA- Flu0rene
~- ~
8o
o
~ ~ 60
.
o:
~_ ~ 4Q
~-
2O
0
I
0
I
8
Concentration
12
16
[-10-5 M]
Fig. 9. Inhibition of endogenous RNase-sensitiveFLV-DNA-polymeraseactivity by tilorone

and congeners
The inhibition of the endogenous activity of FLV-DNA polymerase by tilorone
and congeners is shown in Fig. 9. A maximum inhibition was obtained with DEAPfluoranthene. The inhibitory responses of tilorone (DEAE-fluorenone), DMAAdibenzothiophene and DEAA-fluorene were of the same magnitude; whereas, DMAAdibenzofuran showed a weak response. It is interesting to note that the monosubstituted congener MEAA-fluorene did not inhibit the endogenous reaction at
any concentration.
A similar inhibitory response by the tilorone congeners was exhibited 7) in the
DNA-polymerase system of FLV, catalyzed by the template poly rA-(dT)12. The
143

effect of tilorone and congeners on the FLV-DNA-polymerase reaction, catalyzed
by poly (dA-dT), was also studied 7). This reaction was more sensitive towards
tilorone and its congeners than the endogenous, or poly rA. (dT)l 2-catalyzed
reactions. This is in accordance to our findings on tilorone action, reported
earlier 26).
The data reported above, or elsewhere 7' 26'62)showthat the poly (dA-dT)catalyzed reaction of viral DNA-polymerase is most sensitive to these compounds.
It is possible that the biological activity of these compounds is due to their interaction with the hybrid RNA-DNA (hy.DNA), single-stranded DNA (ss-DNA), or the
DNA-DNA duplex (ds-DNA). It was therefore, interesting to locate the site of action
of tilorone in the viral DNA-polymerase system. It is still not clear whether a
particular site or target in the DNA-polymerase system, other than the true RNAdependent reaction, can be correlated with the biological role of the oncornaviruses.
The key role of the RNA-directed reaction in in-vivo leukemogenesis using purified
enzyme and rifamycin derivatives, has been nicely demonstrated by Wu et a163) .
We have conducted some model studies to analyze the products of the FLVDNA-polymerase reaction under the influence of tilorone. The procedure we
adopted was based on a recent report by Kotler and Becker64) on distamycin A,
which has been shown by us to react with ss-DNA and ds-DNA 6s'66) .
The product analysis of the DNA-polymerase reaction (FLV) in the absence and
in the presence of tilorone (1 x 10 - 4 M) is depicted in Fig. 10. The products of
the viral DNA-polymerase reaction were, under these conditions, eluted in three
species. The first species to be eluted from the column contained ss-DNA, the second
contained the RNA-DNA hybrid.molecules (hy-DNA) and finally, the ds-DNA,
eluted in the last species. Analysis of products synthesized in the presence of tilorone
showed that the ss-DNA and the hybrid species, but not the ds-DNA species were
synthesized. This indicates that tilorone has a low affinity to viral RNA, but can
block the synthesis of ds-DNA by interacting with ss-DNA or hy-DNA.
10. S o m e F u t u r e P r o s p e c t s
The data reported here show that tilorone, a compound endowed with many
interesting biological activities, requires very specific structural parameters for its
biochemical action; and moreover, the same structural entities are also involved in
its interaction with DNA. Using the molecular models of double-stranded helical
DNA, one can examine the intercalation of tilorone at the same molecular dimensions.
This approach could help to find out the groups or substitutions which will anchor
best in A-T rich sites of DNA, thus leading to active derivatives of tilorone.
1 1. A d d e n d u m
Intercalation binding as a molecular mechanism of R factor elimination has been
proposed by Hahn and Ciak 68). Based on earlier data 26-28), that tflorone intercalates
144

2624
22
20
18
%
u
.=_o 14
~
n 10
%
;8
~O4
12
16
20
Fraction no.
24
28
32
Fig. 10. Anatysis of the DNA species synthesized by FLV-DNA polymerase by elution from
hydroxylapatite column. Each column was fiUed with 1 g. of hydroxylapatite and
carefully washed with 0.05 M sodium phosphate (approx. 50 ml). The columns were loaded
with the reaction products, as described in text. The columns were washed with 0.05 M sodium
phosphate buffer, pH 6.8, until equilibrium was reached. Macromolecules were eluted from the
columns by a linear gradient of sodium phosphate (0.05-0.4 M). 9 - - 9 DNA species
synthesized in the absence of tilorone; o - - o, DNA species synthesized in the presence of
10 - 4 M tilorone
into DNA, Hahn 69) studied the effect o f tilorone on the elimination o f resistance
determinants in S. typhimurium. He reported that tilorone at 1 0 - 4 M was able to
eliminate 7 8 - 8 5 % resistance determinants tested against Kanamycin, Chloramphenicol,
Streptomycin and AmpiciUin. Similar observations were made b y De Bary7~
compared the elimination o f four different plasmids ( F ' lac + , R a - l , R I and Rs-a)
by five different compounds (ethidium bromide, sodium dodecyl sulfate, nalidixic
acid, acridine orange and tilorone). Of all these compounds, tilorone hydrochloride
was reported to be most effective eliminator o f plasmids.
Another biological effect o f tilorone, based on its specific interaction to DNA
is a selective inhibition o f sporulation o f B. subtilis strain 60015. At a drug con145

centration of 100 gg/ml the vegetative growth was not inhibited but, the sporulation
was completely blocked 70. The tflorone resistant mutants were asporogenous. The
author has suggested that the selectivity in tilorone action may be due to the fact
that A-T rich regions are involved in the sporulating phase.
146
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148
The volume numbers are printed in italics

Albini, A., and Kisch, H.: Complexation and Activation of Diazenes and Diazo
Compounds by Transition Metals. 65, 105-145 (1976).
Altona, C., and Faber, D. H.: Empirical Force Field Calculations. A Tool in Structural Organic Chemistry. 45, 1-38 (1974).
Anderson, J. E.: Chair-Chair Interconversion of Six-Membered Rings. 45, 139-167
(1974).
Anet, F. A. L.: Dynamics of Eight-Membered Rings in Cyclooctane Class. 45,
169-220 (1974).
Ari~ns, E. J., and Simonis, A.-M.: Design of Bioactive Compounds. 52, 1-61 (1974).
Aurich, H. G. and Weiss, W.: Formation and Reactions of Aminyloxides. 59,
65-111 (1975).
Bardos, T. J.: Antimetabolites: Molecular Design and Mode of Action. 52, 63-98
(1974).
Barnes D. S., see Pettit, L. D.: 28, 85-139 (1972).
Bauer, S. H., and Yokozeki, A.: The Geometric and Dynamic Structures of Fluorocarbons and Related Compounds. 53, 71-119 (1974).
Baumg~rtner, F., and Wiles, D. R.: Radiochemical Transformations and Rearrangements in Organometallic Compounds. 32, 63-108 (1972).
Bernardi, F., see Epiotis, N. D.: 70, 1-242 (1977).
Bernauer, K.: Diastereoisomerism and Diastereoselectivity in Metal Complexes.
65, 1-35 (1976).
Boettcher, R. J., see Mislow, K.: 47, 1-22 (1974).
Brandmiiller, J., and Sehr6tter, H. W.: Laser Raman Spectroscopy of the Solit State.
36, 85-127 (1973).
Bremser, W.: X-Ray Photoelectron Spectroscopy. 36, 1-37 (1973).
Breuer, H.-D., see Winnewisser, G.: 44, 1-81 (1974).
Brewster, J. H.: On the Helicity of Variously Twisted Chains of Atoms. 47, 29-71
(1974).
Brocas, J.: Some Formal Properties of the Kinetics of Pentacoordinate Stereoisomer.
izations. 32, 43-61 (1972).
Brunner, H.: Stereochemistry of the Reactions of Optically Active Organometallic
Transition Metal Compounds. 56, 67-90 (1975).
Buchs, A., see Delfino, A. B.: 39, 109-137 (1973).
Biirger, H., and Eujen, R.: Low-Valent Silicon. 50, 1--41 (1974).
149
Author Index Volumes26-72

Burgermeister, W., and Winkler-Oswatitsch, R.: Complexformation of Monovalent
Cations with Biofunctional Ligands. 69, 91-196 (1977).
Butler, R. S., and deMaine, A. D.: CRAMS - An Automatic Chemical Reaction
Analysis and Modeling System. 58, 39-72 (1975).
Caesar, F.: Computer.Gas Chromatography. 39, 139-167 (1973).
C~rsky, P., and Zaradn~, R.: MO Approach to Electronic Spectra of Radicals. 43,
1-55 (1973).
Chandra, P.: Molecular Approaches for Designing Antiviral and Antitumor
Compounds. 52, 99-139 (1974).
Chandra, P., and Wright, G. J.: Tilorone Hydrochloride, The Drug Profile. 72,
125-148 (1977).
Chapuisat, X., and Jean, Y.: Theoretical Chemical Dynamics: A Tool in Organic
Chemistry. 68, 1-57 (1976).
Cherry, W. R., see Epiotis, N. D.: 70, 1-242 (1977).
Chini, P., and Heaton, B. T.: Tetranuclear Carbonyl Clusters. 71, 1-70 (1977).
Christian, G. D.: Atomic Absorption Spectroscopy for the Determination of
Elements in Medical Biological Samples. 26, 77-112 (1972).
Clark, G. C., see Wasserman, H. H.: 47, 73-156 (1974).
Clerc, T., and Erni, F.: Identification of Organic Compounds by Computer-Aided
Interpretation of Spectra. 39, 91-107 (1973).
Clever, H.: Der Analysenautomat DSA-560.29, 29-43 (1972).
Conner, J. A.: Thermochemical Studies of Organo-Transition Metal Carbonyls and
Related Compounds. 71, 71-110 (1977).
Conners, T. A.: Alkylating Agents. 52, 141-171 (1974).
Craig, D. P., and Mellor, D. P.: Discriminating Interactions Between Chiral Molecules.
63, 1---48 (1976).
Cram, D. J., and Cram, J. M.: Stereochemical Reaction Cycles. 31, 1-43 (1972).
Gresp, T. M., see Sargent, M. V.: 57, 111-143 (1975).
Dauben, W. G., Lodder, G., and Ipaktschi, J.: Photochemistry of 13,~,-Unsaturated
Ketones. 54, 73-114 (1974).
DeClercq, E.: Synthetic Interferon Inducers. 52, 173-198 (1974).
Degens, E. T.: Molecular Mechanisms on Carbonate, Phosphate, and Silica Deposition in the Living Cell. 64, 1-112 (1976).
Delfino, A. B., and Buchs, A.: Mass Spectra and Computers. 39, 109-137 (1973).
DeMaine, A. D., see Butler, R. S.: 58, 39-72 (1975).
DePuy, C. H.: Stereochemistry and Reactivity in Cyclopropane Ring-Cleavageby
Electrophiles. 40, 73-101 (1973).
Devaquet, A.: Quantum-Mechanical Calculations of the Potential Energy Surface of
Triplet States. 54, 1-71 (1974).
Dimroth. K.: Delocalized Phosphorus-Carbon Double Bonds. Phosphamethincyanines,
k3-Phosphorins and ?~s-Phosphorins.38, 1-150 (1973).
D6pp, D.: Reactions of Aromatic Nitro Compounds v/a Excited Triplet States. 55,
49-85 (1975).
Dougherty, R. C.: The Relationship Between Mass Spectrometric. Thermolytic and
Photolytic Reactivity. 45, 93-138 (1974).
150

Dryhurst, G.: Electrochemical Oxidation of Biologically-Important Purines at the
Pyrolytic Graphite Electrode. Relationship to the Biological Oxidation of Purines.
34, 47-85 (1972).
Diirr, H.: Reactivity of Cycloalkene-carbenes. 40, 103-142 (1973).
Dtirr, H.: Triplet Intermediates from Diazo-Compounds (Carbenes). 55, 87-135
(1975).
Diirr, H., and Kober, H.: Triplet States from Azides. 66, 89-114 (1976).
Diirr_ H., and Ruge, B.: Triplet States from Azo Compounds. 66, 53-87 (1976).
Dugundji, J., and Ugi, I.: An Algebraic Model of Constitutional Chemistry as a Basis
for Chemical Computer Programs. 39, 19--64 (I 973).
Eglinton, G., Maxwell, J. R., and Pillinger, C. T.: Carbon Chemistry of the Apollo
Lunar Samples. 44, 83-113 (1974).
Eicher, T., and Weber, J. L.: Structure and Reactivity of Cyclopropenones and
Triafulvenes. 57, 1-109 (1975).
Epiotis, N. D., Cherry, W. R., Shaik, S., Yates, R. L., and Bernardi, F.: Structural
Theory of Organic Chemistry. 70, 1-242 (1977).
Erni, F., see Clerc, T.: 39, 139-167 (1973).
Eujen, R.; see Biirger, H.: 50, 1-41 (1974).
Faber, D. H., see Altona, C.: 45, 1-38 (1974).
Fietzek, P. P., and Kiihn, K.: Automation of the Sequence Analysis by Edman
Degradation of Proteins and Peptides. 29, 1-28 (1972).
Finocchiaro, P., see Mislow, K.: 47, 1-22 (1974).
Fischer, G.: Spectroscopic Implications of Line Broadening in Large Molecules. 66,
115-147 (1976).
Fluck, E.: The Chemistry of Phosphine. 35, 1-64 (1973).
Flygare, W. H., see Sutter, D. H.: 63, 89-196 (1976).
Fowler, F. W., see Gelernter, H.: 41, 113-150 (1973).
Freed, K. F.: The Theory of Raditionless Processes in Polyatomic Molecules. 31,
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Topics in Current Chemistry

Springer-Verlag Berlin Heidelberg New York

Springer-Verlag New York Heidelberg Berlin

Modes of Action of Antimicrobial Agents

Ansamycins: Chemistry, Biosynthesis and Biological Activity

Syntheses and Activity of Heteroprostanoids

Hypolipidaemic Aryloxyacetic Acids

Tilorone Hydrochloride: The Drug Profile

Author Index Volumes 26-72

Department of Chemistry, The University of Texas

Prof. Dr. Klaus Hafner

Institut fiir Organische Chemie der TH

Prof. Dr. Edgar Heilbronner

Physikalisch-Chemisches Institut der Universitiit

Prof. Dr. Sh6 It6

Department of Chemistry, Tohoku University,

Prof. Dr. Jean-Marie Lehn

Institut de Chimie, Universit6 de Strasbourg, 4, rue

Prof. Dr. Kurt Niedenzu

University of Kentucky, College of Arts and Sciences

Prof. Dr. Klaus Schiller

Institut fiir Physikalische Chemie der Universit~it

Prof. Dr. Georg Wittig

Institut fiir Organische Chemie der Universit~it

Springer-Verlag, Postfach 105 280,

Postfach 105 280 - D-6900 Heidelberg 1

175, Fifth Avenue New York, NY 10010

Modes of Action of Antimicrobiai Agents

What Constitutes the Mode o f Action? . . . . . . . . . . . .

HI. The Test Organisms and Drag Effects U p o n Them

IV. The Strategy of Mode of Action Studies

V~ Research on Mechanisms of Action

Modes of Action of Antimicrobial Agents

III. T e s t Organisms and Drug E f f e c t s U p o n T h e m

Modes of Action of Antimicrobial Agents

Modes of Action of Antimicrobial Agents

and streptomycin have no lethal effects on non-growing bacterial suspensions and

One classical and early example of selective inhibition of DNA biosynthesis is

"" ~ " ! J ' "

Modes of Action of Antimicrobial Agents

Modes o f Action of Antimicrobial Agents

Fig, 4. Selective inhibition of protein biosynthesis in E. coli by 1.9 x 10 - 4 M chloramphenicoi39)

4. Inhibitions of Cell Wall Biosynthesis. All bactericidal modes of action involve

Modes of Action of Antimicrobiat Agents

Fig. 6. Sequential phases of

Fig. 7. Fluorescence photomicrograph of Bacillus megaterium after treatment with fluorescent

Modes of Action of Antimicrobial Agents

Modes of Action of Antimicrobial Agents

Modes of Action of AntimicrobialAgents

Received January 18, 1977

Conclusions and Summary

Fig. 1. Schematic structure of the ansamycins

Fig. 2 a. Stereomodel of rifamycin SV. The hydrophobic face

Ansamycins - Chemistry, Biosynthesis and Biological Activity

Fig. 2b. Stereomodel of rifamycin SV. The hydrophilic face

Ansamycins- Chemistry Biosynthesisand BiologicalActivity

Fig. 4. Structural relations between rifamycinsB, O, S and SV

Fig. 5. Structure of rifampicin and its synthesisfrom rifamycin SV

Ctl3 CH3 CHa

CH3 CH3 CH3

CH3 CH3 CH3

Fig. 6. Structure of some naturally occurring rifamycins and halomicin B